CGRP receptor antagonists

ABSTRACT

The disclosures herein relate to novel compounds of formula 
                         
wherein R 1 , R 2 , R 3  and R 4  are as defined herein, and their use in treating, preventing, ameliorating, controlling or reducing cerebrovascular or vascular disorders associated with CGRP receptor function.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.15/713,775, filed Sep. 25, 2017, which is a divisional of U.S. patentapplication Ser. No. 15/336,866, filed Oct. 28, 2016, which claimspriority from GB Patent Application No.: 1519196.8, filed Oct. 30, 2015.The entire contents of these applications are incorporated herein byreference in their entirety.

TECHNICAL FIELD

This application relates to novel compounds and their use as CGRPreceptor antagonists. Compounds described herein may be useful in thetreatment or prevention of cerebrovascular or vascular disorders such asmigraine. The application is also directed to pharmaceuticalcompositions comprising these compounds and the manufacture and use ofthese compounds and compositions in the prevention or treatment of suchcerebrovascular or vascular disorders.

BACKGROUND OF THE INVENTION

Migraine is a highly disabling neurovascular disorder characterized byattacks of moderate to severe headache that are often associated withnausea, vomiting, photophobia, and phonophobia. The attacks can lastfrom 4 to 72 h, and the average attack frequency is 1 or 2 per month.About 20-30% of migraine patients experience transient focal neurologicsymptoms known as aura, which are usually visual and can precede oraccompany the headache. Migraine afflicts about 11% of adults worldwideand results in a significant socioeconomic burden, in terms of bothquality of life and lost productivity.

Whilst the pathomechanism of migraine is still unclear, one of theleading hypotheses is based on activation of the trigeminovascularsystem (TS). Several neuropeptides participate in this activation,calcitonin gene-related peptide (CGRP) playing a crucial role amongthem. CGRP exerts various biological effects through the peripheral andcentral nervous system (CNS). The functional CGRP-receptor (CGRP-R)complex has been well characterized, and novel therapeutic approachestarget CGRP itself and its receptors. This invention relates to thedevelopment of CGRP receptor antagonists (CGRP-RA).

CGRP, a 37-amino acid neuropeptide derived from the gene encodingcalcitonin, is formed from the alternative splicing of thecalcitonin/CGRP gene located on chromosome 11. In humans, CGRP has twoisoforms: α- and β-CGRP. The β-isoform differs from the α-isoform in theamino acids located at positions 3, 22 and 25. The chemical structure ofCGRP involves a disulphide bridge between residues 2 and 7 and anamidated C-terminus. The cyclic cysteine2-cysteine7 motif has a basicrole in receptor activation. In the human trigeminal ganglia (TRIG),CGRP-immunoreactive neurons account for up to 50% of all neurons. It hasbeen demonstrated through an in situ hybridization technique that 40% ofall nerve cell bodies contain CGRP mRNA and CGRP. Double immunostaininghas shown that in the human TRIG CGRP is co-localized with nitric oxidesynthase, substance P (SP), pituitary adenylate cyclase activatingpeptide (PACAP) and nociceptin, which may play a role in thepathomechanism of migraine.

The functional CGRP-R consists of three proteins: i) Calcitonin ReceptorLike Receptor (known as CRLR, CALCRL or CLR) is a seven-transmembranespanning protein, which forms the ligand binding site with; ii) RAMP1,determining the specificity of the receptor; and iii) the CGRP-Rcomponent protein (RCP) couples the receptor to intracellular signaltransduction pathways and to adenylyl cyclase.

It is thought that the C-terminal region of CGRP initially binds to thelarge N-terminal extracellular domain (ECD) of the receptor, likelymaking interactions with both CLR and RAMP1. This initial binding eventgreatly increases the local concentration of the N-terminal region ofCGRP in the vicinity of the juxtamembrane portion of CLR, allowing theirrelatively weak interaction to occur and resulting in receptoractivation. Since mutagenesis experiments indicated that most smallmolecule antagonists interacted with the ECD of CLR/RAMP1, it washypothesized that they bind to this region of the receptor and preventthe initial binding of CGRP to the receptor. A notable exception to thismodel of peptide binding and small molecule receptor antagonism is thehydroxypyridine class of antagonists, which apparently interact withtransmembrane domain 7 (TM7) in CLR and not with the extracellulardomain (Bell I M, J. Med. Chem., 2014, 57(19), 7838-58).

The first clinically tested CGRP-RA, olcegepant, was based on adipeptide backbone, had high molecular weight, and was not orallybioavailable. Nonetheless, when dosed intravenously, olcegepant provedto be an effective antimigraine agent, and this proof-of-concept studygreatly increased interest in the field. Following the success ofolcegepant, a number of orally acting CGRP-RAs were advanced to clinicaltrials. Telcagepant and compounds BI 44370, MK-3207, and BMS-927711 haveall been used for acute treatment of migraine as oral agents. Takentogether, the results from these clinical studies demonstrate thatCGRP-RAs can exhibit similar antimigraine efficacy to the gold standardtriptan drugs but with a significantly lower incidence of adverse eventsthan is typically observed with a triptan. It is worth noting that theavailable data indicate that these CGRP blockers do not causevasoconstriction and suggest that they may have a superiorcardiovascular safety profile to the triptans. One potential concernthat has been reported with some CGRP-RAs is the observation of elevatedlevels of liver transaminases in some patients, and this reportedly ledto the discontinuation of MK-3207. Although elevated liver enzymes werealso found in a small number of subjects after dosing with telcagepantfor an extended period, it is not clear if these findings are in someway mechanism-based or specific to these two compounds. In clinicaltrials for acute migraine therapy, the CGRP-RAs displayed favourableeffects, but their frequent administration was associated with livertoxicity (the elevation of liver transaminases), which limited theirclinical use. Hence, there is a need to develop new CGRP-RAs which donot induce liver injury.

SUMMARY OF THE INVENTION

One possibility to address the risk of liver injury is to target anon-oral route of delivery for a small molecule which will place a lowerburden on the liver through first-pass exposure. The compounds of theinvention can be used for sub-cutaneous, intravenous and/or intranasalroutes of administration. The molecular profile for a CGRP-RA intendedfor such routes of administration differs from the profile required foran oral molecule: extremely high affinity and functional potency,coupled with extremely high solubility is required. Disclosed herein arenovel compounds, and the first medical use of said compounds as CGRPreceptor antagonists.

Compounds of the invention include compounds of formula (I)

or salts thereof, wherein R¹ is selected from H or Q—(C₁-C₆)alkyl; whereQ is a bond, C(O) or C(O)O and where the (C₁-C₆)alkyl can be optionallysubstituted by N(C₁-C₃alkyl)₂ or CO₂H;R² is H or forms a spirocyclic heterocyclic ring with R³;R³ forms a spirocyclic heterocyclic ring with R² or is a heterocyclicring if R² is H; andR⁴ is an optionally substituted aryl group which may be monocyclic orfused to a further ring.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to novel compounds. The invention also relates tothe use of novel compounds as CGRP receptor antagonists. The inventionfurther relates to the use of compounds in the manufacture ofmedicaments for use as CGRP receptor antagonists. The invention furtherrelates to compounds, compositions and medicaments for the treatment ofcerebrovascular or vascular disorders such as migraine (includingsubtypes such as: migraine without aura, chronic migraine, puremenstrual migraine, menstrually-related migraine, migraine with aura,familial hemiplegic migraine, sporadic hemiplegic migraine, basilar-typemigraine, cyclical vomiting, abdominal migraine, benign paroxysmalvertigo of childhood, retinal migraine), status migrainosus, clusterheadache, dialysis headache, paroxysmal hemicrania, osteoarthritis, hotflashes associated with menopause or medically induced menopause due tosurgery or drug treatment, hemicrania continua, cyclic vomitingsyndrome, allergic rhinitis, or rosacea. The invention further relatesto compounds, compositions and medicaments for the treatment of broaderpain states and diseases involving neurogenic inflammation includingdental pain, earache, middle ear inflammation, sunburn, joint painassociated with osteoarthritis and rheumatoid arthritis, cancer pain,fibromyalgia, diabetic neuropathy, pain associated with inflammatorybowel disease—Crohn's disease, gout, complex regional pain syndrome,Behçet's disease, endometriosis pain, back pain or cough.

Compounds exemplified herein are based around the structure:

wherein R¹ is selected from H or Q—(C₁-C₆)alkyl; where Q is a bond, C(O)or C(O)O and where the (C₁-C₆)alkyl can be optionally substituted byN(C₁-C₃alkyl)₂ or CO₂H;R² is H or forms a spirocyclic heterocyclic ring with R³;R³ forms a spirocyclic heterocyclic ring with R² or is a heterocyclicring if R² is H; andR⁴ is an optionally substituted aryl group which may be monocyclic orfused to a further ring.

The optional substituents for R⁴ may be selected from halo, hydroxyl ormethyl. More particularly, the substituent for R⁴ is a substitutedphenyl group wherein the substituents are selected from halo orhydroxyl. In a particular embodiment, R⁴ is a moiety according toformula II

wherein X is halo.

In a more particular embodiment, X is Br.

In a particular embodiment, the substituent for R⁴ is

In a particular embodiment, the substituent for R² is H and R³ isselected from:

In a particular embodiment, R² forms a spirocyclic heterocyclic ringwith R³ to form:

In a particular embodiment, the substituent for R¹ is H, CO₂ ^(t)Bu,CH₂CH₃, CH₂CH₂CH₃, COCH₂CH₂CH₂CH₃, CH₂CH₂N(CH₃)₂ or COCH₂CO₂H. In a moreparticular embodiment, the substituent for R¹ is H.

Compounds of the invention include those of formula (I)

wherein R¹ is selected from H or Q—(C₁-C₆)alkyl; where Q is a bond, C(O)or C(O)O and where the (C₁-C₆)alkyl can be optionally substituted byN(C₁-C₃alkyl)₂ or CO₂H; R² is H or forms a spirocyclic heterocyclic ringwith R³ to form:

and wherein when R² is H, R³ is selected from:

andR⁴ is selected from

In a more particular embodiment, the substituent for R¹ is H.

Further embodiments of the invention include methods of treatmentcomprising administering a compound of formulas (I) as a CGRP receptorantagonist. The treatment using a compound of formulas (I) may be in thetreatment of cerebrovascular or vascular disorders such as migraine(including subtypes such as: migraine without aura, chronic migraine,pure menstrual migraine, menstrually-related migraine, migraine withaura, familial hemiplegic migraine, sporadic hemiplegic migraine,basilar-type migraine, cyclical vomiting, abdominal migraine, benignparoxysmal vertigo of childhood, retinal migraine), status migrainosus,cluster headache, dialysis headache, paroxysmal hemicrania,osteoarthritis, hot flashes associated with menopause or medicallyinduced menopause due to surgery or drug treatment, hemicrania continua,cyclic vomiting syndrome, allergic rhinitis, or rosacea. The inventionfurther relates to compounds, compositions and medicaments for thetreatment of broader pain states and diseases involving neurogenicinflammation including dental pain, earache, middle ear inflammation,sunburn, joint pain associated with osteoarthritis and rheumatoidarthritis, cancer pain, fibromyalgia, diabetic neuropathy, painassociated with inflammatory bowel disease—Crohn's disease, gout,complex regional pain syndrome, Behçet's disease, endometriosis pain,back pain or cough.

Certain novel compounds of the invention show particularly highactivities as CGRP receptor antagonists.

Exemplary compounds include:

The NMR and LCMS properties as well as the biological activities ofthese compounds are set out in Tables 2 and 3.

To the extent that any of the compounds described have chiral centres,the present invention extends to all optical isomers of such compounds,whether in the form of racemates or resolved enantiomers. The inventiondescribed herein relates to all crystal forms, solvates and hydrates ofany of the disclosed compounds however so prepared. To the extent thatany of the compounds and intermediates disclosed herein have acid orbasic centres such as carboxylates or amino groups, then all salt formsof said compounds are included herein. In the case of pharmaceuticaluses, the salt should be seen as being a pharmaceutically acceptablesalt.

Pharmaceutically acceptable salts that may be mentioned include acidaddition salts and base addition salts. Such salts may be formed byconventional means, for example by reaction of a free acid or a freebase form of a compound with one or more equivalents of an appropriateacid or base, optionally in a solvent, or in a medium in which the saltis insoluble, followed by removal of said solvent, or said medium, usingstandard techniques (e.g. in vacuo, by freeze-drying or by filtration).Salts may also be prepared by exchanging a counter-ion of a compound inthe form of a salt with another counter-ion, for example using asuitable ion exchange resin.

Examples of pharmaceutically acceptable salts include acid additionsalts derived from mineral acids and organic acids, and salts derivedfrom metals such as sodium, magnesium, or preferably, potassium andcalcium.

Examples of acid addition salts include acid addition salts formed withacetic, 2,2-dichloroacetic, adipic, alginic, aryl sulfonic acids (e.g.benzenesulfonic, naphthalene-2-sulfonic, naphthalene-1,5-disulfonic andp-toluenesulfonic), ascorbic (e.g. L-ascorbic), L-aspartic, benzoic,4-acetamidobenzoic, butanoic, (+)-camphoric, camphor-sulfonic,(+)-(1S)-camphor-10-sulfonic, capric, caproic, caprylic, cinnamic,citric, cyclamic, dodecylsulfuric, ethane-1,2-disulfonic,ethanesulfonic, 2-hydroxyethanesulfonic, formic, fumaric, galactaric,gentisic, glucoheptonic, gluconic (e.g. D-gluconic), glucuronic (e.g.D-glucuronic), glutamic (e.g. L-glutamic), α-oxoglutaric, glycolic,hippuric, hydrobromic, hydrochloric, hydriodic, isethionic, lactic (e.g.(+)-L-lactic and (±)-DL-lactic), lactobionic, maleic, malic (e.g.(−)-L-malic), malonic, (±)-DL-mandelic, metaphosphoric, methanesulfonic,1-hydroxy-2-naphthoic, nicotinic, nitric, oleic, orotic, oxalic,palmitic, pamoic, phosphoric, propionic, L-pyroglutamic, salicylic,4-amino-salicylic, sebacic, stearic, succinic, sulfuric, tannic,tartaric (e.g.(+)-L-tartaric), thiocyanic, undecylenic and valericacids.

Particular examples of salts are salts derived from mineral acids suchas hydrochloric, hydrobromic, phosphoric, metaphosphoric, nitric andsulfuric acids; from organic acids, such as tartaric, acetic, citric,malic, lactic, fumaric, benzoic, glycolic, gluconic, succinic,arylsulfonic, pamoic acids; and from metals such as sodium, magnesium,or preferably, potassium and calcium.

Also encompassed are any solvates of the compounds and their salts.Preferred solvates are solvates formed by the incorporation into thesolid state structure (e.g. crystal structure) of the compounds of theinvention of molecules of a non-toxic pharmaceutically acceptablesolvent (referred to below as the solvating solvent). Examples of suchsolvents include water, alcohols (such as ethanol, isopropanol andbutanol) and dimethylsulfoxide. Solvates can be prepared byrecrystallising the compounds of the invention with a solvent or mixtureof solvents containing the solvating solvent. Whether or not a solvatehas been formed in any given instance can be determined by subjectingcrystals of the compound to analysis using well known and standardtechniques such as thermogravimetric analysis (TGE), differentialscanning calorimetry (DSC) and X-ray crystallography.

The solvates can be stoichiometric or non-stoichiometric solvates.Particular solvates may be hydrates, and examples of hydrates includehemihydrates, monohydrates and dihydrates.

For a more detailed discussion of solvates and the methods used to makeand characterise them, see Bryn et al., Solid-State Chemistry of Drugs,Second Edition, published by SSCI, Inc of West Lafayette, Ind., USA,1999, ISBN 0-967-06710-3.

“Pharmaceutically functional derivatives” of compounds as defined hereinincludes ester derivatives and/or derivatives that have, or provide for,the same biological function and/or activity as any relevant compound ofthe invention. Thus, for the purposes of this invention, the term alsoincludes prodrugs of compounds as defined herein.

The term “prodrug” of a relevant compound includes any compound that,following oral or parenteral administration, is metabolised in vivo toform that compound in an experimentally-detectable amount, and within apredetermined time (e.g. within a dosing interval of between 6 and 24hours (i.e. once to four times daily)).

Prodrugs of compounds may be prepared by modifying functional groupspresent on the compound in such a way that the modifications arecleaved, in vivo when such prodrug is administered to a mammaliansubject. The modifications typically are achieved by synthesizing theparent compound with a prodrug substituent. Prodrugs include compoundswherein a hydroxyl, amino, sulfhydryl, carboxyl or carbonyl group in acompound is bonded to any group that may be cleaved in vivo toregenerate the free hydroxyl, amino, sulfhydryl, carboxyl or carbonylgroup, respectively.

Examples of prodrugs include, but are not limited to, esters andcarbamates of hydroxyl functional groups, ester groups of carboxylfunctional groups, N-acyl derivatives and N-Mannich bases. Generalinformation on prodrugs may be found e.g. in Bundegaard, H. “Design ofProdrugs” p. 1-92, Elsevier, New York-Oxford (1985).

Definitions

C₁-C₆ Alkyl

Alkyl means an aliphatic hydrocarbon group. The alkyl group may bestraight or branched. “Branched” means that at least one carbon branchpoint is present in the group, for example isopropyl or tertiarybutyl.C₁-C₃ alkyl groups include methyl, ethyl, n-propyl, i-propyl. The alkylgroup may be optionally substituted.

Heterocyclic

Heterocyclic means a cyclic group which may be aromatic in which atleast one ring member is other than carbon. For example, at least onering member (for example one, two or three ring members) may be selectedfrom nitrogen, oxygen and sulphur. The point of attachment of heteroarylgroups may be via any atom of the ring system. Exemplary heteroarylgroups include pyridyl, indazolyl,1,4-dihydro-2H-pyrido[2,3-d][1,3]oxazin-2-one,1,3-dihydro-2H-imidazo[4,5-b]pyridin-2-one,3,4-dihydroquinazolin-2(1H)-one, quinolin-2(1H)-one, piperidinyl,piperazinyl, and the like.

Optionally Substituted

“Optionally substituted” as applied to any group means that the saidgroup may if desired be substituted with one or more substituents, whichmay be the same or different.

The term “pharmaceutical composition” in the context of this inventionmeans a composition comprising an active agent and comprisingadditionally one or more pharmaceutically acceptable carriers. Thecomposition may further contain ingredients selected from, for example,diluents, adjuvants, excipients, vehicles, preserving agents, fillers,disintegrating agents, wetting agents, emulsifying agents, suspendingagents, sweetening agents, flavouring agents, perfuming agents,antibacterial agents, antifungal agents, lubricating agents anddispersing agents, depending on the nature of the mode of administrationand dosage forms. The compositions may take the form, for example, oftablets, dragees, powders, elixirs, syrups, liquid preparationsincluding suspensions, sprays, inhalants, tablets, lozenges, emulsions,solutions, cachets, granules, capsules and suppositories, as well asliquid preparations for injections, including liposome preparations.

The dosages may be varied depending upon the requirements of thepatient, the severity of the condition being treated, and the compoundbeing employed. Determination of the proper dosage for a particularsituation is within the skill of the art. Generally, treatment isinitiated with the smaller dosages which are less than the optimum doseof the compound. Thereafter the dosage is increased by small incrementsuntil the optimum effect under the circumstances is reached. Forconvenience, the total daily dosage may be divided and administered inportions during the day if desired.

The magnitude of an effective dose of a compound will, of course, varywith the nature of the severity of the condition to be treated and withthe particular compound and its route of administration. The selectionof appropriate dosages is within the ability of one of ordinary skill inthis art, without undue burden. In general, the daily dose range may befrom about 10 μg to about 30 mg per kg body weight of a human andnon-human animal, preferably from about 50 μg to about 30 mg per kg ofbody weight of a human and non-human animal, for example from about 50μg to about 10 mg per kg of body weight of a human and non-human animal,for example from about 100 μg to about 30 mg per kg of body weight of ahuman and non-human animal, for example from about 100 μg to about 10 mgper kg of body weight of a human and non-human animal and mostpreferably from about 100 μg to about 1 mg per kg of body weight of ahuman and non-human animal.

Preparation of the Compounds of the Invention

Compounds of the invention may be prepared by procedures including thosein Scheme 1. Details of many of the standard transformations such asthose in the routes below and others which could be used to perform thesame transformations can be found in standard reference textbooks suchas “Organic Synthesis”, M. B. Smith, McGraw-Hill (1994) or “AdvancedOrganic Chemistry”, 4^(th) edition, J. March, John Wiley & Sons (1992).

Urea formations between amino acid intermediates, for example methylesters of amino acids, and amine intermediates can be formed underconditions using a coupling agent such as DSC or CDI in the presence ofa base such as triethylamine or DIPEA in solvents such as DMF and/orDCM. The methyl ester portion of the subsequently formed ureaderivatives can be saponified using aqueous bases such as lithiumhydroxide or sodium hydroxide in a suitable solvent such as THF, MeOH,1,4-dioxane, EtOAc or a mixture thereof. The acid intermediates thusformed can be converted into amide examples under standard conditions,for example using a coupling agent such as HATU or HBTU, in the presenceof a base such as DIPEA in a suitable solvent such as DMF. Alternativelyan acid chloride can be coupled with an amine to yield an amide, in thepresence of a base such as Et₃N, in a suitable solvent such as DCM. Theamine partners for such amide couplings can be prepared using anappropriate combination of standard transformations (for examplereductive aminations using an amine, an aldehyde or ketone, and areducing agent such as sodium triacetoxyborohydride in a solvent such asDCM in the presence of acetic acid; or amide formation under conditionssuch as those detailed above) and removal of standard protecting groupsunder conditions which can be found in reference textbooks, for example“Protecting Groups”, 3^(rd) edition, P. J. Kocieński, Georg ThiemeVerlag (2005). One such transformation is the removal of atert-butoxycarbonyl group (commonly known as a Boc group) from an amineunder acidic conditions such as HCl in a solvent such as 1,4-dioxane,MeOH, EtOH, DCM or combinations thereof. It can be appreciated that Bocdeprotection of amine intermediates of the invention which possessadditional basic centres may result in hydrochloride salts of differentstoichiometries. For example the Boc deprotection of an intermediatewith one additional basic centre will result in the formation of a newamine intermediate which is for example the mono-hydrochloride ordi-hydrochloride salt, which will often be used without neutralisationof the hydrochloride salt to produce the free base of the intermediate,as it can be appreciated that in the subsequent amide formation anexcess of a base such as DIPEA or triethylamine is typically used toneutralise the hydrochloride salt. Amine intermediates of the inventionformed by Boc-deprotection which are used without neutralisation to thefree base are named herein as the hydrochloride (×HCl), and the presentinvention extends to all salt forms of the said intermediates. Anothersuch protecting group removal is the deprotection of acarbobenzyloxy-protected amine (commonly known as a Cbz or Z group)using reductive conditions such as catalysis by palladium on carbon in asolvent such as EtOH in the presence of gaseous H₂ or by using acommercially available hydrogenation reactor which combinescontinuous-flow chemistry with in-situ hydrogen generation (for examplean H-Cube hydrogenation reactor, ThalesNano Nanotechnology Inc.,Budapest, Hungary). Alternative conditions for the removal of aCbz-protecting group include transfer hydrogenation, for example using apalladium on carbon catalyst in the presence of ammonium formate orcyclohexa-1,4-diene, or both ammonium formate and cyclohexa-1,4-diene,in a solvent such as EtOH or aqueous EtOH at an elevated temperaturesuch as 70° C.

General Procedures

Where no preparative routes are included, the relevant intermediate iscommercially available. Commercial reagents were utilized withoutfurther purification. Room temperature (rt) refers to approximately20-27° C. ¹H NMR spectra were recorded at 400 MHz on Bruker, Varian orJEOL instruments. Chemical shift values are expressed in parts permillion (ppm), i.e. (δ)-values. The following abbreviations are used forthe multiplicity of the NMR signals: s=singlet, br=broad, d=doublet,t=triplet, q=quartet, quin=quintet, h=heptet, dd=doublet of doublets,dt=double of triplets, m=multiplet. Coupling constants are listed as Jvalues, measured in Hz. NMR and mass spectroscopy results were correctedto account for background peaks. Chromatography refers to columnchromatography performed using silica and executed under positivepressure (flash chromatography) conditions. LCMS experiments werecarried out using electrospray conditions under the conditions below.LCMS data are given in the format: Mass ion, electrospray mode (positiveor negative), retention time (experimental text and Table 1); Mass ion,electrospray mode (positive or negative), retention time, approximatepurity (Table 2).

Method A. Instruments: Hewlett Packard 1100 with G1315A DAD, MicromassZQ; Column: Waters X-Bridge C-18, 2.5 micron, 2.1×20 mm or PhenomenexGemini-NX C-18, 3 micron, 2.0×30 mm; Gradient [time (min)/solvent D in C(%)]: 0.00/2, 0.10/2, 8.40/95, 10.00/95; Solvents: solvent C=2.5 LH₂O+2.5 mL 28% ammonia in water solution; solvent D=2.5 L MeCN+135 mLH₂O+2.5 mL 28% ammonia in water solution; Injection volume 1 μL; UVdetection 230 to 400 nM; column temperature 45° C.; Flow rate 1.5mL/min.

Method B. Instruments: Agilent Technologies 1260 Infinity LC withChemstation software, Diode Array Detector, Agilent 6120B SingleQuadrupole MS with API-ES Source; Column: Phenomenex Gemini-NX C-18, 3micron, 2.0×30 mm; Gradient [time (min)/solvent D in C (%)]: 0.00/5,2.00/95, 2.50/95, 2.60/5, 3.00/5; Solvents C and D are as describedabove in Method A; Injection volume 0.5 μL; UV detection 190 to 400 nM;column temperature 40° C.; Flow rate 1.5 mL/min.

Method C. As detailed for method A, except with Gradient [time(min)/solvent D in C (%)]: 0.00/2, 0.10/2, 2.50/95, 3.50/95.

Method D. Instruments: Acquity UPLC coupled with SQD mass spectrometer;Column: Acquity UPLC BEH C18, 1.7 micron, 2.1×50 mm; Gradient [time(min)/solvent B in A (%)]: 0.00/3, 1.50/100, 1.90/100, 2.00/3; Solvents:solvent A=10 mM aqueous solution of NH₄HCO₃ (adjusted to pH 10 withammonia); solvent B=MeCN; Injection volume 1 μL; UV detection 210 to 350nM; column temperature 40° C.; Flow rate 0.9 mL/min.

ABBREVIATIONS

CDI=1,1′-carbonyldiimidazole

DCM=dichloromethane

DIPEA=N,N-diisopropylethylamine

DMAC=N,N-dimethylacetamide

DMF=dimethylformamide

DSC=N,N′-disuccinimidyl carbonate

DMSO=dimethylsulfoxide

ES=electrospray

EtOAc=ethyl acetate

h=hour(s)

HATU=1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium3-oxid hexafluorophosphate

HBTU=N,N,N′,N′-tetramethyl-O-(1H-benzotriazol-1-yl)uroniumhexafluorophosphate

L=litre

LC=liquid chromatography

LCMS=liquid chromatography mass spectrometry

MeCN=acetonitrile

min=minute(s)

MS=mass spectrometry

NMR=nuclear magnetic resonance

rcf=relative centrifugal force

rpm=revolutions per minute

rt=room temperature

s=second(s)

TFA=trifluoroacetic acid

THF=tetrahydrofuran

Prefixes n-, s-, i-, t- and tert- have their usual meanings: normal,secondary, iso, and tertiary.

Synthesis of Intermediates

Preparation of Carboxylic Acid Intermediates

Typical procedure for the preparation of carboxylic acid intermediatesvia urea formation and subsequent saponification, as exemplified by thepreparation of Intermediate 7,(2R)-3-(7-methyl-1H-indazol-5-yl)-2-{[(2′-oxo-1′,2′-dihydro-1H-spiro[piperidine-4,4′-pyrido[2,3-d][1,3]oxazin]-1-yl)carbonyl]amino}propanoicacid.

Step 1) Et₃N (2.26 mL, 16.3 mmol) was added to a solution of (R)-methyl2-amino-3-(7-methyl-1H-indazol-5-yl) propanoate dihydrochloride(Intermediate 5, 995 mg, 3.3 mmol) and DSC (917 mg, 3.6 mmol) in DMF (20mL) and the mixture stirred at rt for 30 min.Spiro[piperidine-4,4′-[4H]pyrido[2,3-d][1,3]oxazin]-2′(1′H)-one(Intermediate 4, 785 mg, 3.6 mmol) was then added portionwise and thereaction mixture stirred at rt for 18 h before concentration in vacuo.The residue was partitioned between H₂O and MeOH/DCM (1:9), the phaseswere separated and the aqueous layer was washed with H₂O. Residual solidfrom the separation step was dissolved in MeOH and the combined organiclayers were concentrated in vacuo and purified by flash chromatography,eluting with EtOAc in MeOH (20:1), to yield methyl(2R)-3-(7-methyl-1H-indazol-5-yl)-2-{[(2′-oxo-1′,2′-dihydro-1H-spiro[piperidine-4,4′-pyrido[2,3-d][1,3]oxazin]-1-yl)carbonyl]amino}propanoate (1.06 g, 2.22 mmol)as a white solid.

LCMS (Method A): m/z 479.3 (ES+), at 2.61 min, 100%.

¹H NMR: (400 MHz, DMSO-d₆) δ: 1.59-1.75 (m, 2H), 1.78-1.90 (m, 2H), 2.45(s, 3H), 2.90-3.08 (m, 4H), 3.59 (s, 3H), 3.86-3.96 (m, 2H), 4.28-4.38(m, 1H), 6.94-7.06 (m, 3H), 7.32 (dd, J=7.4, 1.2, 1H), 7.39 (s, 1H),7.95 (s, 1H), 8.18 (dd, J=5.1, 1.6, 1H), 10.79 (s, 1H), 13.04 (s, 1H).

Step 2) Methyl(2R)-3-(7-methyl-1H-indazol-5-yl)-2-{[(2′-oxo-1′,2′-dihydro-1H-spiro[piperidine-4,4′-pyrido[2,3-d][1,3]oxazin]-1-yl)carbonyl]amino}propanoate(1.06 g, 2.22 mmol) was dissolved in THF (15 mL) and MeOH (3 mL) and anaqueous solution of LiOH (1 M, 4.4 mL, 4.4 mmol) was added dropwise.After stirring at rt for 3.5 h further aqueous LiOH (1 M, 2.2 mL, 2.2mmol) was added dropwise and the mixture stirred for 1 h at rt beforeconcentration under a stream of nitrogen. The residue was dissolved in aminimum volume of H₂O and cooled to 0° C. Aqueous 1 M HCl was addeddropwise to adjust the pH to ≤3 and the resulting precipitate wasisolated by filtration, washed with cold H₂O and Et₂O to yield the titlecompound (877 mg, 1.89 mmol) as a pale yellow solid.

Data in Table 1.

Intermediate 8,(2R)-3-(7-methyl-1H-indazol-5-yl)-2-({[4-(2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-1-yl)piperidin-1-yl]carbonyl}amino)propanoic acid

The title compound (1.50 g, 3.2 mmol) was prepared over two steps from(R)-methyl 2-amino-3-(7-methyl-1H-indazol-5-yl)propanoate (Intermediate5, 1.00 g, 4.3 mmol) and1-(piperidin-4-yl)-1,3-dihydro-2H-imidazo[4,5-b]pyridin-2-one(Intermediate 1, 1.02 g, 4.7 mmol) using the methods of Intermediate 7.

Data in Table 1.

Intermediate 14,3,5-dibromo-N-[(2′-oxo-1′,2′-dihydro-1H-spiro[piperidine-4,4′-pyrido[2,3-d][1,3]oxazin]-1-yl)carbonyl]-D-tyrosine

The title compound (561 mg, 1.0 mmol) was prepared over two steps from3,5-dibromo-D-tyrosine methyl ester (Intermediate 6, 530 mg, 1.5 mmol)and spiro[piperidine-4,4′-[4H]pyrido[2,3-d][1,3]oxazin]-2′(1′H)-one(Intermediate 4, 362 mg, 1.7 mmol) using the methods of Intermediate 7.

Data in Table 1.

Intermediate 11,3,5-dibromo-N-{[4-(2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-1-yl)piperidin-1-yl]carbonyl}-D-tyrosine

The title compound (214 mg, 0.37 mmol) was prepared over two steps from3,5-dibromo-D-tyrosine methyl ester (Intermediate 6, 357 mg, 1.01 mmol)and 1-(piperidin-4-yl)-1,3-dihydro-2H-imidazo[4,5-b]pyridin-2-one(Intermediate 1, 362 mg, 1.42 mmol) using the methods of Intermediate 7.

Data in Table 1.

Intermediate 12,3,5-dibromo-N-{[4-(2-oxo-1,4-dihydroquinazolin-3(2H)-yl)piperidin-1-yl]carbonyl}-D-tyrosine

The title compound (224 mg, 0.38 mmol) was prepared over two steps from3,5-dibromo-D-tyrosine methyl ester (Intermediate 6, 353 mg, 1.00 mmol)and 3-(piperidin-4-yl)-3, 4-dihydroquinazolin-2(1H)-one (Intermediate 3,254 mg, 1.10 mmol) using the methods of Intermediate 7.

Data in Table 1.

Intermediate 13,(2R)-3-(7-methyl-1H-indazol-5-yl)-2-({[4-(2-oxo-1,4-dihydroquinazolin-3(2H)-yl)piperidin-1-yl]carbonyl}amino)propanoic Acid

The title compound (561 mg, 1.18 mmol) was prepared over two steps from(R)-methyl 2-amino-3-(7-methyl-1H-indazol-5-yl)propanoate (Intermediate5, 917 mg, 3.93 mmol) and3-(piperidin-4-yl)-3,4-dihydroquinazolin-2(1H)-one (Intermediate 3, 1.00g, 4.32 mmol) using the methods of Intermediate 7.

Data in Table 1.

Intermediate 10,3,5-dibromo-N-{[4-(2-oxo-1,2-dihydroquinolin-3-yl)piperidin-1-yl]carbonyl}-D-tyrosine

The title compound (77 mg, 0.13 mmol) was prepared over two steps from3,5-dibromo-D-tyrosine methyl ester (Intermediate 6, 103 mg, 0.29 mmol)and 3-(piperidin-4-yl) quinolin-2(1H)-one (Intermediate 2, 73 mg, 0.32mmol) using the methods of Intermediate 7.

Data in Table 1.

Intermediate 9,(2R)-3-(7-methyl-1H-indazol-5-yl)-2-({[4-(2-oxo-1,2-dihydroquinolin-3-yl)piperidin-1-yl]carbonyl}amino)propanoic Acid

Step 1) To a solution of (R)-methyl 2-amino-3-(7-methyl-1H-indazol-5-yl)propanoate (Intermediate 5, 6.05 g, 25.9 mmol) in DMF (60 mL) under N₂at approximately −20° C. was added CDI (8.40 g, 51.8 mmol) and themixture was stirred for 15 mins while keeping the temperature below −10°C. A solution of H₂O (2.34 mL) in a few mL of DMF was added and stirringcontinued for 15 mins while keeping the temperature below −10° C.3-(Piperidin-4-yl) quinolin-2(1H)-one (Intermediate 2, 6.99 g, 30.6mmol), DIPEA (4.93 mL, 28.2 mmol) and DCM (20 mL) were then added inthat order and the mixture was heated to 40° C. under N₂ for 12 h. Aftercooling to rt, 2 M HCl (aq) (38.7 mL) was added and the mixture wasextracted twice with DCM. The combined organic extracts were washedthree times with H₂O, dried (Na₂SO₄) and concentrated in vacuo.Purification by flash chromatography, eluting with MeOH/DCM (5:95),yielded methyl(2R)-3-(7-methyl-1H-indazol-5-yl)-2-({[4-(2-oxo-1,2-dihydroquinolin-3-yl)piperidin-1-yl]carbonyl}amino)propanoate (10.4 g, 21.3 mmol) as a lighttan solid.

¹H NMR: (400 MHz, CDCl₃) δ: 1.40-1.60 (m, 2H), 1.95-1.97 (m, 2H), 2.46(s, 3H), 2.90-3.00 (m, 2H), 3.11-3.26 (m, 3H), 3.76 (s, 3H), 4.07-4.12(m, 2H), 4.86-4.91 (m, 1H), 5.18 (d, J=7.6, 1H), 6.93 (s, 1H), 7.17-7.21(m, 1H), 7.24 (s, 1H), 7.32 (s, 1H), 7.43-7.54 (m, 3H), 7.95 (s, 1H),10.70 (s, 2H).

Step 2) To a solution of methyl(2R)-3-(7-methyl-1H-indazol-5-yl)-2-({[4-(2-oxo-1,2-dihydroquinolin-3-yl)piperidin-1-yl]carbonyl}amino)propanoate (9.79 g, 20.1 mmol) in1,4-dioxane (150 mL) was added a solution of LiOH·H₂O (1.26 g, 30.0mmol) in H₂O (150 mL) and the mixture was stirred at rt for 2 h. Thereaction mixture was concentrated in vacuo to near-dryness andre-dissolved in H₂O before being acidified with aqueous 2 M HCl(approximately 15 mL) whilst being rapidly stirred. The resulting thickwhite precipitate was isolated by filtration and washed with H₂O untilthe washings were near neutral pH. Drying in vacuo yielded the titlecompound (8.11 g, 17.1 mmol) as an off-white solid.

Data in Table 1.

Preparation of Amine Intermediates

Intermediate 17, tert-butyl4-{(2S)-2-amino-3-oxo-3-[4-(pyridin-4-yl)piperazin-1-yl]propyl}piperidine-1-carboxylate

Step 1) To a solution of tert-butyl4-[(2S)-2-{[(benzyloxy)carbonyl]amino}-3-methoxy-3-oxopropyl]piperidine-1-carboxylate(Intermediate 16, 4.29 g, 10.2 mmol) in 1,4-dioxane (50 mL) was added asolution of LiOH·H₂O (856 mg, 20.4 mmol) in H₂O (50 mL) and the mixturewas stirred for 5 h. The reaction mixture was concentrated in vacuo tonear-dryness and re-dissolved in H₂O before being acidified with 0.5 MNaHSO₄ (aq). The resulting thick white precipitate was extracted intoEtOAc and the combined organic layers were washed with brine, dried(MgSO₄) and concentrated in vacuo to yieldN-[(benzyloxy)carbonyl]-3-[1-(tert-butoxycarbonyl)piperidin-4-yl]-L-alanine as a white foam (4.01 g, 9.87 mmol).

¹H NMR: (400 MHz, DMSO-d₆) δ: 0.85-1.07 (m, 2H), 1.39 (s, 9H), 1.45-1.68(m, 5H), 2.55-2.71 (m, 2H), 3.86-3.94 (m, 1H), 4.85-4.98 (m, 2H), 5.04(s, 2H), 7.28-7.39 (m, 5H), 7.53 (d, J=8.3, 1H), 12.5 (br s, 1H).

Step 2) To a solution ofN-[(benzyloxy)carbonyl]-3-[1-(tert-butoxycarbonyl)piperidin-4-yl]-L-alanine (4.00 g, 9.84 mmol) in DMF (80 mL) was addedHBTU (4.10 g, 10.81 mmol) followed by DIPEA (3.74 mL, 21.47 mmol) and1-(4-pyridyl) piperazine (Intermediate 15, 1.69 g, 10.35 mmol) and themixture was stirred at rt for 3 h. The reaction mixture was concentratedin vacuo to near-dryness and the residue dissolved in EtOAc, washedtwice with H₂O, twice with NaHCO₃ (aq) and brine, dried (Na₂SO₄) andconcentrated in vacuo. Purification by flash chromatography, elutingwith MeOH/DCM (5:95), yielded tert-butyl4-{(2S)-2-{[(benzyloxy)carbonyl]amino}-3-oxo-3-[4-(pyridin-4-yl)piperazin-1-yl]propyl}piperidine-1-carboxylate(4.51 g, 8.18 mmol) as an off-white foam.

¹H NMR: (400 MHz, CDCl₃) δ: 1.07-1.16 (br m, 2H), 1.44 (s, 9H),1.54-1.60 (br m, 2H), 1.88-1.91 (m, 1H), 2.62-2.65 (br m, 2H), 3.47-3.65(br m, 6H), 3.81-3.86 (br m, 2H), 4.05 (br m, 2H), 4.26 (br m, 2H),4.70-4.74 (m, 1H), 5.04-5.12 (m, 2H), 5.70 (d, J=8.8, 1H), 6.82 (d,J=6.0, 2H), 7.26-7.34 (m, 5H), 8.10 (br s, 2H).

Step 3) A solution of tert-butyl4-{(2S)-2-{[(benzyloxy)carbonyl]amino}-3-oxo-3-[4-(pyridin-4-yl)piperazin-1-yl]propyl}piperidine-1-carboxylate (557 mg, 1.01 mmol) inEtOH (20 mL) was eluted five times through a Pd/C cartridge at 50° C.using a continuous flow hydrogenation reactor (H-Cube, ThalesNanoNanotechnology Inc., Budapest, Hungary) in the presence of H₂ (full H₂mode), monitoring conversion to desired product by LCMS. Upon >95%conversion the reaction mixture was concentrated in vacuo to yield thetitle compound (455 mg, 1.09 mmol) as a yellow glassy solid.

Data in Table 1.

Intermediate 18, methylN-[(benzyloxy)carbonyl]-3-(1-propylpiperidin-4-yl)-L-alaninate

Step 1) HCl in 1,4-dioxane (4 M, 10 mL, 40 mmol) was added to a solutionof tert-butyl4-[(2S)-2-{[(benzyloxy)carbonyl]amino}-3-methoxy-3-oxopropyl]piperidine-1-carboxylate(Intermediate 16, 1.00 g, 2.38 mmol) in MeOH (10 mL). After stirring atrt for 2 h the reaction mixture was concentrated in vacuo to yieldmethyl N-[(benzyloxy)carbonyl]-3-piperidin-4-yl-L-alaninatehydrochloride (850 mg) which was used without purification in thesubsequent step.

LCMS (Method C): m/z 321.2 (ES⁺) at 1.66 min.

Step 2) A mixture of methylN-[(benzyloxy)carbonyl]-3-piperidin-4-yl-L-alaninate hydrochloride (500mg, 1.40 mmol), propionaldehyde (120 μL, 1.68 mmol) and glacial aceticacid (96 μL, 1.68 mmol) in DCM (10 mL) was stirred at rt for 1 h, beforethe addition of sodium triacetoxyborohydride (356 mg, 1.68 mmol). Afterstirring overnight at rt the mixture was concentrated in vacuo andpurified by flash column chromatography, eluting with 0-10% MeOH in DCMto yield the title compound (400 mg, 1.10 mmol) as a colourless oil.

Data in Table 1.

Intermediate 19,(2S)-2-amino-3-(1-propylpiperidin-4-yl)-1-[4-(pyridin-4-yl)piperazin-1-yl]propan-1-one

Step 1) Aqueous sodium hydroxide (1 M, 5 mL, 5.0 mmol) was added to asolution of methylN-[(benzyloxy)carbonyl]-3-(1-propylpiperidin-4-yl)-L-alaninate(Intermediate 18, 400 mg, 1.10 mmol) in MeOH (5 mL). After stirring atrt overnight the reaction mixture was partially concentrated in vacuo toremove MeOH and acidified to pH≤2 with 1 M aqueous HCl. Concentration invacuo yielded crudeN-[(benzyloxy)carbonyl]-3-(1-propylpiperidin-4-yl)-L-alanine (385 mg)which was used without purification in the subsequent step.

LCMS (Method B): m/z 349.0 (ES⁺) at 0.79 min.

Step 2) A mixture of crudeN-[(benzyloxy)carbonyl]-3-(1-propylpiperidin-4-yl)-L-alanine (385 mg),1-(4-pyridyl)piperazine (Intermediate 15, 215 mg, 1.32 mmol), HATU (505mg, 1.33 mmol) and DIPEA (383 μL, 2.20 mmol) in DMF (5 mL) was stirredat rt for 3 h. Concentration in vacuo yielded benzyl{(2S)-1-oxo-3-(1-propylpiperidin-4-yl)-1[4-(pyridin-4-yl)piperazin-1-yl]propan-2-yl}carbamate (542 mg) as a pale orange, viscousoil which was used without purification in the subsequent step.

LCMS (Method B): m/z 493.9 (ES⁺) at 1.45 min.

Step 3) Ammonium formate (643 mg, 11.0 mmol) was added to a solution ofbenzyl{(2S)-1-oxo-3-(1-propylpiperidin-4-yl)-1-[4-(pyridin-4-yl)piperazin-1-yl]propan-2-yl}carbamate(crude, 542 mg) in EtOH (40 mL) and H₂O (10 mL). Palladium on carbon(10%, 10 mg) was added and the mixture was heated at 70° C. under N₂overnight. After cooling to rt the mixture was filtered through celiteand the filtrate concentrated in vacuo to yield the title compound as ayellow viscous oil (115 mg) which was used without purification in theformation of Example 4.

Data in Table 1.

Intermediate 20,1-(4-{(2S)-2-amino-3-oxo-3-[4-(pyridin-4-yl)piperazin-1-yl]propyl}piperidin-1-yl)pentan-1-one

Step 1) HCl in 1,4-dioxane (4 M, 2.30 mL, 9.20 mmol) was added to asolution of tert-butyl4-{(2S)-2-{[(benzyloxy)carbonyl]amino}-3-oxo-3-[4-(pyridin-4-yl)piperazin-1-yl]propyl}piperidine-1-carboxylate(Intermediate 17, Step 2 product) (1.30 g, 2.30 mmol) in EtOAc (23 mL)and the mixture was stirred at rt overnight. After concentration invacuo purification by flash column chromatography eluting withDCM/MeOH/7N NH₃ (90:5:5) in MeOH yielded benzyl{(2S)-1-oxo-3-(piperidin-4-yl)-1-[4-(pyridin-4-yl)piperazin-1-yl]propan-2-yl}carbamate(740 mg, 1.64 mmol).

LCMS (Method B): m/z 452.2 (ES⁺) at 1.30 min.

Step 2) A mixture of pentanoic acid (137 mg, 0.36 mmol), benzyl{(2S)-1-oxo-3-(piperidin-4-yl)-1-[4-(pyridin-4-yl)piperazin-1-yl]propan-2-yl}carbamate (146 mg, 0.32 mmol), triethylamine(209 μL, 1.50 mmol) and HATU (137 mg, 0.36 mmol) in DMF (3 mL) wasstirred at rt overnight. 1 M aqueous sodium carbonate solution (20 mL)was added, the mixture was concentrated in vacuo and purified bygradient flash column chromatography, eluting with 0-10% (1:1 MeOH/7NNH₃ in MeOH) in DCM, to yield benzyl{(2S)-1-oxo-3-(1-pentanoylpiperidin-4-yl)-1-[4-(pyridin-4-yl)piperazin-1-yl]propan-2-yl}carbamate.

LCMS (Method B): m/z 536.2 (ES⁺) at 1.38 min.

Step 3) A solution of benzyl{(2S)-1-oxo-3-(1-pentanoylpiperidin-4-yl)-1-[4-(pyridin-4-yl)piperazin-1-yl]propan-2-yl}carbamate in EtOH was eluted through a Pd/Ccartridge at 50° C. using a continuous flow hydrogenation reactor(H-Cube, ThalesNano Nanotechnology Inc., Budapest, Hungary) in thepresence of H₂ (full H₂ mode), monitoring conversion to desired productby LCMS. Upon >95% conversion the reaction mixture was concentrated invacuo to yield the title compound which was used in its entirety(assumed to be 0.32 mmol) without purification in the formation ofExample 6.

Data in Table 1.

Intermediate 21,(2S)-2-amino-3-(1-ethylpiperidin-4-yl)-1-[4-(pyridin-4-yl)piperazin-1-yl]propan-1-one

The title compound (286 mg, 0.83 mmol) was prepared over four steps frommethyl N-[(benzyloxy)carbonyl]-3-piperidin-4-yl-L-alaninatehydrochloride (Intermediate 18, Step 1 product) (320 mg, 0.90 mmol) andacetaldehyde (62 μL, 1.10 mmol) using the methods of Intermediates 18and 19.

Data in Table 1.

Intermediate 22,(2S)-2-amino-3-{1-[2-(dimethylamino)ethyl]piperidin-4-yl}-1-[4-(pyridin-4-yl)piperazin-1-yl]propan-1-one

The title compound (306 mg, used crude without purification in theformation of Example 12) was prepared over four steps from methylN-[(benzyloxy)carbonyl]-3-piperidin-4-yl-L-alaninate hydrochloride(Intermediate 18, Step 1 product) (1.0 g, 2.81 mmol) and2-(dimethylamino)acetaldehyde sulfite (568 mg, 3.36 mmol) using themethods of Intermediates 18 and 19.

Data in Table 1.

Intermediate 23, ethyl3-(4-{(2S)-2-amino-3-oxo-3-[4-(pyridin-4-yl)piperazin-1-yl]propyl}piperidin-1-yl)-3-oxopropanoate

Step 1) HCl in 1,4-dioxane (4 M, 10.0 mL, 40.0 mmol) was added to asolution of tert-butyl4-{(2S)-2-{[(benzyloxy)carbonyl]amino}-3-oxo-3-[4-(pyridin-4-yl)piperazin-1-yl]propyl}piperidine-1-carboxylate(Intermediate 17, Step 2 product) (600 mg, 1.09 mmol) in MeOH (10 mL)and the mixture was stirred at rt overnight. Concentration in vacuoyielded benzyl{(2S)-1-oxo-3-(piperidin-4-yl)-1-[4-(pyridin-4-yl)piperazin-1-yl]propan-2-yl}carbamatedihydrochloride (570 mg, 1.09 mmol) as a sticky white solid.

LCMS (Method B): m/z 452.2 (ES⁺) at 1.72 min.

Step 2) Ethyl 3-chloro-3-oxopropanoate (151 μL, 1.20 mmol) was added toa solution of Et₃N (608 μL, 4.36 mmol) and benzyl{(2S)-1-oxo-3-(piperidin-4-yl)-1-[4-(pyridin-4-yl)piperazin-1-yl]propan-2-yl}carbamate dihydrochloride (570 mg, 1.09 mmol)in DCM (20 mL) and the mixture stirred at rt overnight. Afterconcentration in vacuo purification by gradient flash columnchromatography, eluting with 0-10% MeOH in DCM yielded the desiredmaterial (ethyl3(4-{(2S)-2-{[(benzyloxy)carbonyl]amino}-3-oxo-3-[4-(pyridin-4-yl)piperazin-1-yl]propyl}piperidin-1-yl)-3-oxopropanoate,pale yellow sticky solid, 510 mg), as an approximate 2:1 mixture withbis-acylated byproduct (ethyl3-(4-{(2S)-2-{[(benzyloxy)carbonyl](3-ethoxy-3-oxopropanoyl)amino}-3-oxo-3-[4-(pyridin-4-yl)piperazin-1-yl]propyl}piperidin-1-yl)-3-oxopropanoate).The mixture was used in the following step without further purification.

LCMS (Method B): m/z 566.2 (ES⁺) at 1.18 min (desired material); m/z680.2 (ES⁺) at 0.79 min (bis-acylated byproduct).

Step 3) A mixture of crude ethyl3-(4-{(2S)-2-{[(benzyloxy)carbonyl]amino}-3-oxo-3-[4-(pyridin-4-yl)piperazin-1-yl]propyl}piperidin-1-yl)-3-oxopropanoate (510 mg) andcyclohexa-1,4-diene (0.85 mL, 9.00 mmol) in EtOH (20 mL) was flushedwith N₂ before the addition of 10% Pd/C (10 mg). After heating at 70° C.for 1 h, the reaction mixture was cooled to rt and ammonium formate (568mg, 9.00 mmol) was added. The mixture was heated at 70° C. under N₂overnight before cooling to rt and filtering. The filtrate wasconcentrated in vacuo to yield the crude title compound (194 mg) whichwas used without purification in the formation of Example 13.

Data in Table 1.

TABLE 1 Intermediates Intermediate Name Data 11-(piperidin-4-yl)-1,3-dihydro-2H- Commercially available, CAS No.185961-99-3 imidazo[4,5-b]pyridin-2-one 23-(piperidin-4-yl)quinolin-2(1H)- Commercially available, CAS No.205058-78-2 one 3 3-(piperidin-4-yl)-3,4- Commercially available, CASNo. 79098-75-2 dihydroquinazolin-2(1H)-one 4spiro[piperidine-4,4′-[4H]pyrido[2,3- Commercially available, CAS No.753440-87-8 d][1,3]oxazin]-2′(1′H)-one 5 (R)-methyl 2-amino-3-(7-methyl-Commercially available, 1H-indazol-5-yl)propanoate CAS No. 890044-58-3(free base), CAS No. 1414976-14-9 (dihydrochloride salt) 63,5-dibromo-D-tyrosine methyl Commercially available, CAS No.173383-29-4 ester 7 (2R)-3-(7-methyl-1H-indazol-5- LCMS (Method A): m/z463.5 (ES−), 465.3 yl)-2-{[(2′-oxo-1′,2′-dihydro-1H- (ES+), at 0.10 min.¹H NMR (400 MHz, DMSO- spiro[piperidine-4,4′-pyrido[2,3- d6) δ:1.53-1.91 (m, 4H), 2.44 (s, 3H), 2.89-3.14 d][1,3]oxazin]-1- (m, 5H),3.89 (t, J = 11.5, 2H), 4.23 (br s, 1H), yl)carbonyl]amino}propanoicacid 6.73 (d, J = 7.8, 1H), 6.93-7.06 (m, 2H), 7.31 (d, J = 7.4, 1H),7.38 (s, 1H), 7.93 (s, 1H), 8.17 (dd, J = 5.1, 1.2, 1H), 10.78 (s, 1H),13.00 (br s, 1H) 8 (2R)-3-(7-methyl-1H-indazol-5- LCMS (Method A): m/z464.1 (ES+), at 1.14 yl)-2-({[4-(2-oxo-2,3-dihydro-1H- min. ¹H NMR (400MHz, DMSO-d₆) δ: 1.62- imidazo[4,5-b]pyridin-1- 1.67 (m, 2H), 1.87-2.12(m, 2H), 2.38-2.52 (m, yl)piperidin-1- 1H), 2.46 (s, 3H), 2.70-2.80 (m,2H), 2.98 (dd, yl]carbonyl}amino)propanoic acid J = 13.7, 9.8, 1H), 3.09(dd, J = 13.7, 4.3, 1H), 4.08 (br d, J = 12.9, 2H), 4.20-4.27 (m, 1H),4.28-4.38 (m, 1H), 6.75 (d, J = 8.2, 1H), 6.88 (dd, J = 7.8, 5.5, 1H),7.42 (s, 1H), 7.27 (d, J = 7.8, 1H), 7.42 (s, 1H), 7.88 (dd, J = 5.1,1.2, 1H), 7.96 (s, 1H), 11.54 (br s, 1H), 12.99 (br s, 1H) 9(2R)-3-(7-methyl-1H-indazol-5- LCMS (Method A): m/z 474.3 (ES+), at 1.82yl)-2-({[4-(2-oxo-1,2- min. ¹H NMR (400 MHz, DMSO-d₆) δ: 1.25-dihydroquinolin-3-yl)piperidin-1- 1.36 (m, 2H), 1.72-1.78 (m, 2H), 2.48(s, 3H), yl]carbonyl}amino)propanoic acid 2.66-2.78 (m, 2H), 2.88-2.94(m, 1H), 2.97-3.03 (m, 1H), 3.10 (dd, J = 8.4, 3.4, 1H), 4.08 (d, J =12.0, 2H), 4.24-4.30 (m, 1H), 6.57 (d, J = 8.0, 1H), 7.04 (s, 1H), 7.15(dd, J = 12.4, 1.2, 1H), 7.27 (d, J = 8.4, 1H), 7.41-7.45 (m, 2H), 7.54(s, 1H), 7.62 (dd, J = 6.8, 1.2, 1H), 7.97 (s, 1H), 11.69 (s, 1H),12.1-13.1 (br s, 2H). 10 3,5-dibromo-N-{[4-(2-oxo-1,2- LCMS (Method A):m/z 592.0, 594.0, 596.0 dihydroquinolin-3-yl)piperidin-1- (ES+), at 0.14min. ¹H NMR (400 MHz, DMSO- yl]carbonyl}-D-tyrosine d₆) δ: 1.14-1.40 (m,2H), 1.75-1.77 (m, 2H), 2.68-2.99 (m, 5H), 3.97-4.19 (m, 3H), 6.71 (d, J= 8.2 Hz, 1H), 7.14 (t, J = 7.6 Hz, 1H), 7.26 (t, J = 7.4, 1H),7.36-7.49 (m, 3H), 7.53-7.70 (m, 2H), 9.74 (s, 1H), 11.77 (s, 1H), 12.54(br s, 1H) 11 3,5-dibromo-N-{[4-(2-oxo-2,3- LCMS (Method A): m/z 580.3,582.1, 584.0 dihydro-1H-imidazo[4,5-b]pyridin- (ES−), 582.1, 584.1,586.1 (ES+), at 0.39 min. ¹H 1-yl)piperidin-1-yl]carbonyl}-D- NMR (400MHz, DMSO-d₆) δ: 1.53-1.76 (d, tyrosine J = 11.3, 2H), 1.91-2.21 (m,2H), 2.72-2.89 (m, 3H), 2.97 (dd, J = 13.7, 4.3, 1H), 4.10 (d, J = 12.5,2H), 4.15-4.26 (m, 1H), 4.38 (t, J = 12.3, 1H), 6.82 (d, J = 8.2, 1H),6.96 (dd, J = 7.8, 5.1, 1H), 7.34 (d, J = 7.8, 1H), 7.47 (s, 2H), 7.89(d, J = 4.3, 1H), 9.72 (br s, 1H), 11.57 (br s, 1H) (1 exchangeableproton not observed) 12 3,5-dibromo-N-{[4-(2-oxo-1,4- LCMS (Method A):m/z 593.2, 595.3, 597.1 dihydroquinazolin-3(2H)- (ES−), 595.1, 597.1,599.1 (ES+), at 0.41 min. ¹H yl)piperidin-1-yl]carbonyl}-D- NMR (400MHz, DMSO-d₆) δ: 1.41-1.68 (m, tyrosine 4H), 2.65 (t, J = 11.7, 1H),2.70-2.96 (m, 3H), 3.80-3.95 (m, 2H), 4.01 (d, J = 12.9, 1H), 4.23 (s,2H), 4.24-4.38 (m, 1H), 6.24 (d, J = 4.7, 1H), 6.74 (d, J = 7.8, 1H),6.84 (t, J = 7.4, 1H), 7.02-7.16 (m, 2H), 7.24 (s, 2H), 9.21 (s, 1H) (2exchangeable protons not observed) 13 (2R)-3-(7-methyl-1H-indazol-5-LCMS (Method A): m/z 475.4 (ES−), 477.3 yl)-2-({[4-(2-oxo-1,4- (ES+), at0.66 min. ¹H NMR (400 MHz, DMSO- dihydroquinazolin-3(2H)- d6) δ:1.36-1.66 (m, 4H), 2.47 (s, 3H), 2.59-2.78 yl)piperidin-1- (m, 2H),2.92-3.14 (m, 3H), 4.00 (t, J = 16.0, 2H), yl]carbonyl}amino)propanoicacid 4.06-4.20 (m, 2H), 4.20-4.33 (m, 1H), 6.47 (br s, 1H), 6.75 (d, J =7.8, 1H), 6.86 (t, J = 7.4, 1H), 7.01 (s, 1H), 7.06-7.17 (m, 2H), 7.36(s, 1H), 7.96 (s, 1H), 9.21 (s, 1H), 12.99 (s, 1H) (1 exchangeableproton not observed) 14 3,5-dibromo-N-[(2′-oxo-1′,2′- LCMS (Method A):m/z 583.3, 585.0, 587.0 dihydro-1H-spiro[piperidine-4,4′- (ES+), at 0.13min. ¹H NMR (400 MHz, DMSO- pyrido[2,3-d][1,3]oxazin]-1- d₆) δ:1.63-2.01 (m, 4H), 2.82 (t, J = 10.9, 1H), yl)carbonyl]-D-tyrosine2.90-3.14 (m, 3H), 3.95 (t, J = 12.3, 2H), 4.15- 4.28 (m, 1H), 6.86 (d,J = 8.2, 1H), 7.08 (dd, J = 7.2, 5.3, 1H), 7.45 (s, 2H), 7.55 (d, J =7.4, 1H), 8.20 (d, J = 4.7, 1H), 9.75 (br s, 1H), 10.83 (s, 1H) (1exchangeable proton not observed) 15 1-(4-pyridinyl)piperazineCommercially available, CAS No. 1008-91-9 16 tert-butyl 4-[(2S)-2-Commercially available, CAS No. 195877-54-4{[(benzyloxy)carbonyl]amino}-3- methoxy-3-oxopropyl]piperidine-1-carboxylate 17 tert-butyl 4-{(2S)-2-amino-3-oxo-3- LCMS (Method A): m/z318.3, 362.2, 418.3 [4-(pyridin-4-yl)piperazin-1- (ES+), at 2.70 min. ¹HNMR (400 MHz, DMSO- yl]propyl}piperidine-1-carboxylate d₆) δ: 0.89-1.09(m, 2H), 1.38 (s, 9H), 1.43-1.69 (m, 5H), 1.80 (d, J = 10.9, 1H), 2.67(br s, 2H), 3.42-3.79 (m, 7H), 3.84-4.00 (m, 2H), 4.11-4.25 (m, 1H),4.34 (br s, 2H), 6.84 (d, J = 6.6, 2H), 8.19 (d, J = 6.2, 2H) 18 methylN-[(benzyloxy)carbonyl]-3- LCMS (Method B): m/z 363.0 (ES+), at 1.57(1-propylpiperidin-4-yl)-L- min. ¹H NMR (400 MHz, CDCl₃) δ: ppm 0.95 (t,alaninate J = 7.4, 3H), 1.47-1.62 (m, 2H), 1.70-1.80 (m, 6H), 2.01-2.07(m, 1H), 2.36-2.46 (m, 2H), 2.69- 2.76 (m, 2H), 3.36-3.43 (m, 2H), 3.73(s, 3H), 4.33-4.43 (m, 1H), 5.09-5.12 (m, 2H), 5.31-5.33 (m, 1H),7.30-7.38 (m, 5H) 19 (2S)-2-amino-3-(1-propylpiperidin- LCMS (Method B):m/z 360.2 (ES+), at 0.97 4-yl)-1-[4-(pyridin-4-yl)piperazin- min.1-yl]propan-1-one 20 1-(4-{(2S)-2-amino-3-oxo-3-[4- LCMS (Method B): m/z402.2 (ES+), at 1.04 (pyridin-4-yl)piperazin-1- min.yl]propyl}piperidin-1-yl)pentan-1- one 21(2S)-2-amino-3-(1-ethylpiperidin- LCMS (Method B): m/z 346.2 (ES+), at1.03 4-yl)-1-[4-(pyridin-4-yl)piperazin- min. 1-yl]propan-1-one 22(2S)-2-amino-3-{1-[2- LCMS (Method B): m/z 389.2 (ES+), at 0.81(dimethylamino)ethyl]piperidin-4- min.yl}-1-[4-(pyridin-4-yl)piperazin-1- yl]propan-1-one 23 ethyl3-(4-{(2S)-2-amino-3-oxo-3- LCMS (Method B): m/z 432.2 (ES+), at 0.85[4-(pyridin-4-yl)piperazin-1- min. yl]propyl}piperidin-1-yl)-3-oxopropanoateSynthesis of Examples

Typical procedures for the preparation of examples via amide coupling,and where appropriate, deprotection, as exemplified by the preparationof the below examples.

Procedure 1:

Example 11N-[(2R)-3-(7-methyl-1H-indazol-5-yl)-1-oxo-1-({(2S)-1-oxo-3-(piperidin-4-yl)-1-[4-(pyridin-4-yl)piperazin-1-yl]propan-2-yl}amino)propan-2-yl]-2′-oxo-1′,2′-dihydro-1H-spiro[piperidine-4,4′-pyrido[2,3-d][1,3]oxazine]-1-carboxamide

Step 1: HATU (4.57 g, 12.0 mmol) was added to a solution of(2R)-3-(7-methyl-1H-indazol-5-yl)-2-{[(2′-oxo-1′,2′-dihydro-1H-spiro[piperidine-4,4′-pyrido [2,3-d][1,3]oxazin]-1-yl)carbonyl]amino}propanoic acid (Intermediate 7, 4.65 g, 10.0 mmol) in DMF(150 mL), followed after 15 min by the addition of tert-butyl4-{(2S)-2-amino-3-oxo-3-[4-(pyridin-4-yl)piperazin-1-yl]propyl}piperidine-1-carboxylate (Intermediate 17, 4.60 g,11.0 mmol) and DIPEA (6.86 mL, 40.1 mmol). The mixture was stirred at rtfor 17 h before the addition of H₂O (600 mL). The resulting precipitatewas isolated by filtration, washed with H₂O, and dissolved in a smallamount of MeOH. Co-evaporation twice with toluene yielded crudetert-butyl4-{(2S)-2-{[(2R)-3-(7-methyl-1H-indazol-5-yl)-2-{[(2′-oxo-1′,2′-dihydro-1H-spiro[piperidine-4,4′-pyrido[2,3-d][1,3]oxazin]-1-yl)carbonyl]amino}propanoyl]amino}-3-oxo-3-[4-(pyridin-4-yl)piperazin-1-yl]propyl}piperidine-1-carboxylate (7.82 g) which was usedin the next step without further purification. A second batch ofmaterial (3.53 g) was prepared using this method.

LCMS (Method D): m/z 864.7 (ES⁺) at 0.88 min.

Step 2: TFA (31 mL) was added to a solution of tert-butyl4-{(2S)-2-{[(2R)-3-(7-methyl-1H-indazol-5-yl)-2-{[(2′-oxo-1′,2′-dihydro-1H-spiro[piperidine-4,4′-pyrido[2,3-d][1,3]oxazin]-1-yl)carbonyl]amino}propanoyl]amino}-3-oxo-3-[4-(pyridin-4-yl)piperazin-1-yl]propyl}piperidine-1-carboxylate(Step 1, Batch 1, 7.82 g) in DCM (150 mL) and the solution stirred at rtfor 1 h. Toluene (50 mL) was added and the mixture was concentrated invacuo. DCM (200 mL) and H₂O (50 mL) were added and the pH adjusted toapproximately 12 with 2 M (aq) NaOH solution. The phases were separatedand the aqueous layer extracted with DCM/isopropanol (1:1, 5×200 mL).The combined organic phases were concentrated in vacuo. Purification bygradient flash column chromatography, eluting with 0-100% (DCM/MeOH/7NNH₃ in MeOH (4:1:0.4)) in DCM yielded the title compound. A second batchof title compound was prepared using this method from Step 1, Batch 2material. The purified title compound from both batches was combined,dissolved in a mixture of DCM, MeOH and diisopropyl ether, sonicated andconcentrated in vacuo to yield the title compound (5.30 g, 6.94 mmol).

Data in Table 2.

Example 143,5-dibromo-Nα-[(2′-oxo-1′,2′-dihydro-1H-spiro[piperidine-4,4′-pyrido[2,3-d][1,3]oxazin]-1-yl)carbonyl]-N-{(2S)-1-oxo-3-(piperidin-4-yl)-1-[4-(pyridin-4-yl)piperazin-1-yl]propan-2-yl}-D-tyrosinamide

Step 1) A solution of HATU (7.84 g, 20.6 mmol) and3,5-dibromo-N-[(2′-oxo-1′,2′-dihydro-1H-spiro[piperidine-4,4′-pyrido[2,3-d][1,3]oxazin]-1-yl)carbonyl]-D-tyrosine(Intermediate 14, 10.0 g, 17.2 mmol) in DMF (75 mL) was stirred at rtfor 30 min before the addition of tert-butyl4-{(2S)-2-amino-3-oxo-3-[4-(pyridin-4-yl)piperazin-1-yl]propyl}piperidine-1-carboxylate(Intermediate 17, 7.72 g, 18.5 mmol) and DIPEA (11.8 mL, 68.8 mmol). Thereaction mixture was stirred at rt overnight before cold H₂O (500 mL),saturated aqueous NaHCO₃ and DCM (200 mL) were added. The phases wereseparated and the aqueous phase was extracted with DCM (3×200 mL). Thecombined organic phases were washed with brine (200 mL), concentrated invacuo, and co-evaporated with toluene. Purification by gradient flashcolumn chromatography, eluting with 0-100% solvent B in DCM (wheresolvent B=DCM/MeOH/7N NH₃ in MeOH (90:9:1.5) yielded3,5-dibromo-Nα-{(2S)-3-[1-(tert-butoxycarbonyl)piperidin-4-yl]-1-oxo-1-[4-(pyridin-4-yl)piperazin-1-yl]propan-2-yl}-N-[(2′-oxo-1′,2′-dihydro-1H-spiro[piperidine-4,4′-pyrido[2,3-d][1,3]oxazin]-1-yl)carbonyl]-D-tyrosinamide (9.7 g, 9.86 mmol) asa white solid.

LCMS (Method D): m/z 984.5 (ES⁺) at 0.78 min.

Step 2:3,5-Dibromo-Nα-{(2S)-3-[1-(tert-butoxycarbonyl)piperidin-4-yl]-1-oxo-1-[4-(pyridin-4-yl)piperazin-1-yl]propan-2-yl}-Nα-[(2′-oxo-1′,2′-dihydro-1H-spiro[piperidine-4,4′-pyrido[2,3-d][1,3]oxazin]-1-yl)carbonyl]-D-tyrosinamide (9.7 g, 9.86 mmol) wasdissolved in DCM (70 mL), cooled to 0° C., and TFA (15 mL) was addeddropwise. The mixture was stirred at rt for 10 min before the additionof toluene (50 mL) and concentration in vacuo. DCM (200 mL) and H₂O (100mL) were added, the pH was adjusted to approximately 10 with 2N aqueousNaOH solution. The resulting precipitate was isolated by filtration,dissolved in DCM/MeOH (1:1, 300 mL), concentrated in vacuo, andco-evaporated several times with MeOH and toluene. Trituration fromMeOH/methyl tert-butyl ether yielded the title compound (5.30 g, 6.00mmol).

Data in Table 2.

Procedure 2:

Example 1N-[(2R)-3-(7-methyl-1H-indazol-5-yl)-1-oxo-1-({(2S)-1-oxo-3-(piperidin-4-yl)-1-[4-(pyridin-4-yl)piperazin-1-yl]propan-2-yl}amino)propan-2-yl]-4-(2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-1-yl)piperidine-1-carboxamide

Step 1: DIPEA (0.12 mL, 0.66 mmol) was added to a solution of HATU (99mg, 0.22 mmol),(2R)-3-(7-methyl-1H-indazol-5-yl)-2-({[4-(2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-1-yl)piperidin-1-yl]carbonyl}amino)propanoicacid (Intermediate 8, 100 mg, 0.22 mmol) and tert-butyl4-{(2S)-2-amino-3-oxo-3-[4-(pyridin-4-yl)piperazin-1-yl]propyl}piperidine-1-carboxylate(Intermediate 17, 92 mg, 0.22 mmol) in DMF (2 mL) and the reactionmixture was stirred at rt for 10 d before concentration in vacuo toyield crude tert-butyl4-{(2S)-2-{[(2R)-3-(7-methyl-1H-indazol-5-yl)-2-({[4-(2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-1-yl)piperidin-1-yl]carbonyl}amino)propanoyl]amino}-3-oxo-3-[4-(pyridin-4-yl)piperazin-1-yl]propyl}piperidine-1-carboxylate(190 mg, viscous brown oil) which was used without purification in thesubsequent step.

LCMS (Method C): m/z 863.5 (ES⁺) at 1.89 min.

Step 2: Crude tert-butyl4-{(2S)-2-{[(2R)-3-(7-methyl-1H-indazol-5-yl)-2-({[4-(2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-1-yl)piperidin-1-yl]carbonyl}amino)propanoyl]amino}-3-oxo-3-[4-(pyridin-4-yl)piperazin-1-yl]propyl}piperidine-1-carboxylate(190 mg) was dissolved in MeOH (5 mL) and HCl in dioxane (4 M, 5.0 mL,20.0 mmol) was added. The reaction mixture was stirred at rt for 2 hbefore concentration in vacuo. Purification by preparative reversedphase HPLC (Phenomenex Gemini-NX 5 μm C18 column, 100×30 mm, elutingwith 15 to 90% MeCN/Solvent B over 26 min at 30 mL/min [where solvent Bis 0.2% of (28% NH₃/H₂O) in H₂O] and collecting fractions by monitoringat 205 nm) yielded Example 1 (25 mg, 0.03 mmol) as a beige solid.

Data in Table 2.

Procedure 3:

Example 133-(4-{(2S)-2-{[(2R)-3-(7-methyl-1H-indazol-5-yl)-2-({[4-(2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-1-yl)piperidin-1-yl]carbonyl}amino)propanoyl]amino}-3-oxo-3-[4-(pyridin-4-yl)piperazin-1-yl]propyl}piperidin-1-yl)-3-oxopropanoic Acid, Ammonium Salt

Step 1: A mixture of DIPEA (0.27 mL, 1.52 mmol), HATU (172 mg, 0.45mmol),(2R)-3-(7-methyl-1H-indazol-5-yl)-2-({[4-(2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-1-yl) piperidin-1-yl]carbonyl}amino)propanoic acid(Intermediate 8, 176 mg, 0.38 mmol) and ethyl3-(4-{(2S)-2-amino-3-oxo-3-[4-(pyridin-4-yl)piperazin-1-yl]propyl}piperidin-1-yl)-3-oxopropanoate(Intermediate 23, 194 mg, 0.45 mmol) in DMF (10 mL) was stirred at rtovernight before concentration in vacuo. The crude material was filteredthrough a short plug of SiO₂, eluting with MeOH, before furtherpurification by preparative HPLC (Phenomenex Gemini-NX 5 μm C18 column,100×30 mm, eluting with 15 to 35% MeCN/Solvent B over 12.5 min at 30mL/min [where solvent B is 0.2% of (28% NH₃/H₂O) in H₂O], collectingfractions by monitoring at 205 nm). During the purification, cleavage ofthe ethyl ester was observed. Carboxylic acid containing fractions werecombined and added to 10 mL of concentrated aqueous ammonia, and themixture was allowed to stand at rt overnight before concentration invacuo to yield the title compound (10 mg, 0.01 mmol) as a colourlesssolid.

Data in Table 2.

Further examples prepared by the above procedures are detailed in Table2.

TABLE 2 Ex. Intermediates/ LCMS data No. Name Procedure ¹H NMR (MethodA) 1 N-[(2R)-3-(7-methyl-1H- 8, 17 (400 MHz, CD₃OD) δ: ppm 1.02- m/z763.2 indazol-5-yl)-1-oxo-1- Procedure 2 1.32 (m, 3H), 1.34-1.61 (m,3H), (ES+), at 1.10 ({(25)-1-oxo-3-(piperidin- 1.66-1.84 (m, 3H),1.86-1.93(m, min, 95% 4-yl)-1-[4-(pyridin-4- 1H), 1.96-2.10 (m, 1H),2.17-2.26 yl)piperazin-1-yl]propan- (m, 1H), 2.43-2.53 (m, 2H), 2.70-2-yl}amino)propan-2-yl]- 2.73 (m, 1H), 2.80-2.93 (m, 2H),4-(2-oxo-2,3-dihydro-1H- 2.99-3.08 (m, 2H), 3.10-3.24 (m,imidazo[4,5-b]pyridin-1- 2H), 3.25-4.42 (m, 1H), 3.44-3.56yl)piperidine-1- (m, 4H), 3.60-3.80 (m, 3H), 3.84- carboxamide 3.93 (m,2H), 4.11-4.15 (m, 2H), 4.44-4.36 (m, 1H, 4.54-4.57 (m, 1H), 6.80-6.87(m, 2H), 6.96 (dd, J = 8.0, 5.3, 1H), 7.16 (s, 1H), 7.31 (d, J = 7.8,1H), 7.48 (s, 1H), 7.92 (m, 1H), 7.97 (s, 1H), 8.08-8.15 (m, 2H) (5exchangeable protons not observed) 2 tert-butyl 4-{(2S)-2- 9, 17 (400MHz, DMSO-d₆) δ: ppm 0.73- m/z 871.5 (ES⁻), {[(2R)-3-(7-methyl-1H-Procedure 1, 1.04 (m, 2H), 1.08-1.40 (m, 2H), 873.5 (ES⁺), atindazol-5-yl)-2-({[4-(2- Step 1 1.40-1.54 (m, 9H), 1.55-1.80 (m, 2.54min, 96% oxo-1,2-dihydroquinolin- 6H), 2.45 (s, 3H), 2.57-2.75 (m,3-yl)piperidin-1- 4H), 2.77-2.93 (m, 2H), 2.93-3.07yl]carbonyl}amino)propanoyl]amino}- (m, 1H), 3.09-3.30 (m, 4H), 3.36-3-oxo-3-[4- 3.59 (m, 4H), 3.61-3.93 (m, 3H), (pyridin-4-yl)piperazin-1-4.05 (d, J = 12.5, 2H), 4.27-4.49 (m, yl]propyl}piperidine-1- 1H), 4.82(q, J = 7.3, 1H), 6.58 (d, carboxylate J = 8.6, 1H), 6.77 (d, J = 6.2,2H), 7.13 (t, J = 8.2, 2H), 7.25 (d, J = 8.2, 1H), 7.35-7.53 (m, 3H),7.60 (d, J = 7.8, 1H), 7.95 (s, 1H), 8.12 (d, J = 5.9, 2H), 8.33 (d, J =9.0, 1H), 11.75 (s, 1H), 13.00 (s, 1H) 3 N-[(2R)-3-(7-methyl-1H- 9, 17(400 MHz, DMSO-d₆) δ: ppm 1.00- m/z 771.6 (ES⁻), indazol-5-yl)-1-oxo-1-Procedure 1 1.32 (m, 4H), 1.32-1.43 (m, 1H), 773.5 (ES⁺), at({(2S)-1-oxo-3-(piperidin- 1.43-1.57 (m, 2H), 1.57-1.78 (m, 2.88 min,96% 4-yl)-1-[4-(pyridin-4- 3H), 1.84 (d, J = 13.7, 1H), 2.52 (s,yl)piperazin-1-yl]propan- 3H), 2.60-2.78 (m, 4H), 2.78-3.072-yl}amino)propan-2-yl]- (m, 3H), 3.07-3.27 (m, 3H), 3.75- 4-(2-oxo-1,2-3.93 (m, 6H), 3.93-4.14 (m, 2H), dihydroquinolin-3- 4.17-4.45 (m, 1H),4.81 (q, J = 7.8, yl)piperidine-1- 1H), 6.59 (d, J = 8.2, 1H), 7.00-7.22carboxamide (m, 4H), 7.25 (d, J = 7.8, 1H), 7.35- 7.55 (m, 3H), 7.59 (d,J = 7.4, 1H), 7.97 (s, 1H), 8.07-8.37 (m, 3H), 8.42 (d, J = 8.6, 2H),11.75 (s, 1H), 13.40 (br s, 1H) 4 N-[(2R)-3-(7-methyl-1H- 9, 19 (400MHz, CD₃OD) δ: ppm 0.89 (t, m/z 815.6 indazol-5-yl)-1-oxo-1- Procedure1, J = 7.4, 3H), 0.98-1.21 (m, 2H), (ES+), at 3.32 ({(2S)-1-oxo-3-(1-Step 1 1.27-1.36 (m, 1H), 1.37-1.52 (m, min, 95%propylpiperidin-4-yl)-1-[4- 5H), 1.57-1.67 (m, 3H), 1.76-1.86(pyridin-4-yl)piperazin-1- (m, 2H), 2.01 (s, 3H), 2.18-2.22 (m,yl]propan-2- 2H), 2.46-2.53 (m, 2H), 2.76-2.79 yl}amino)propan-2-yl]-4-(m, 1H), 2.80-2.90 (m, 2H), 2.98- (2-oxo-1,2- 3.06 (m, 2H), 3.16-3.22(m, 1H), dihydroquinolin-3- 3.34 (s, 3H), 3.37-3.40 (m, 1H),yl)piperidine-1- 3.43-3.55 (m, 3H), 3.59-3.65 (m, carboxamide 1H),3.84-3.91 (m, 2H), 4.07-4.16 (m, 2H), 4.63 (t, J = 7.8, 1H), 6.79- 6.81(m, 2H), 7.14 (s, 1H), 7.21- 7.23 (m, 1H), 7.25-7.32 (m, 1H), 7.45-7.48(m, 2H), 7.55 (s, 1H), 7.62-7.64 (m, 1H), 7.97 (s, 1H), 8.08-8.10 (m,2H) (4 exchangeable protons not observed) 5 3,5-dibromo-Na-{[4-(2- 10,17  (400 MHz, MeOD) δ: ppm 1.25- m/z 891.4, oxo-1,2-dihydroquinolin-Procedure 1 1.54 (m, 5H), 1.54-1.69 (m, 2H), 893.2, 895.33-yl)piperidin-1- 1.80-1.93 (m, 3H), 2.04 (d, J = 13.3, (ES⁺), at 2.20yl]carbonyl}-N-{(2S)-1- 1H), 2.76-3.09 (m, 7H), 3.34-3.43 and 2.44 min,oxo-3-(piperidin-4-yl)-1- (m, 3H), 3.52-3.63 (m, 1H), 3.63- 100%[4-(pyridin-4-yl)piperazin- 3.79 (m, 3H), 3.80-4.04 (m, 4H),1-yl]propan-2-yl}-D- 4.04-4.21 (m, 2H), 4.43 (t, J = 7.8, tyrosinamide1H), 7.14 (d, J = 7.4, 2H), 7.25 (t, J = 7.6, 1H), 7.32 (d, J = 8.2,1H), 7.44 (s, 2H), 7.50 (t, J = 7.4, 1H), 7.64-7.79 (m, 2H), 8.14 (d, J= 7.4, 2H) (5 exchangeable protons not observed) 6N-[(2R)-3-(7-methyl-1H- 9, 20 (400 MHz, DMSO-d₆) δ: 0.78-1.11 m/z 857.7(ES⁺), indazol-5-yl)-1-oxo-1- Procedure 1, (m, 6H), 1.14-1.61 (m, 13H),1.63- at 1.76 min, ({(2S)-1-oxo-3-(1- Step 1 1.82 (m, 3H), 2.12-2.34 (m,3H), 100% pentanoylpiperidin-4-yl)- 2.63-3.10 (m, 7H), 3.35-3.85 (m,1-[4-(pyridin-4- 7H), 3.90-4.48 (m, 4H), 4.70-4.91yl)piperazin-1-yl]propan- (m, 1H), 6.52-6.91 (m, 3H), 7.06-2-yl}amino)propan-2-yl]- 7.33 (m, 3H), 7.36-7.69 (m, 4H), 4-(2-oxo-1,2-7.95 (s, 1H), 8.09-8.15 (m, 2H), dihydroquinolin-3- 8.34 (d, J = 8.9,1H), 11.58-11.94 yl)piperidine-1- (m, 1H), 12.81-13.16 (m, 1H)carboxamide 7 N-[(2R)-1-({(2S)-3-(1- 9, 21 (400 MHz, CD₃OD) δ: ppm 1.00-m/z 801.6 ethylpiperidin-4-yl)-1-oxo- Procedure 1, 1.22 (m, 5H),1.23-1.35 (m, 2H), (ES+), at 1.88, 1-[4-(pyridin-4- Step 1 1.39-1.54 (m,4H), 1.64-1.91 (m, 95% yl)piperazin-1-yl]propan- 5H), 2.38-2.45 (m, 2H),2.55 (s, 2-yl}amino)-3-(7-methyl- 3H), 2.79-3.08 (m, 7H), 3.11-3.251H-indazol-5-yl)-1- (m, 2H), 3.35-3.43 (m, 1H), 3.47-oxopropan-2-yl]-4-(2-oxo- 3.69 (m, 4H), 3.83-3.91 (m, 2H),1,2-dihydroquinolin-3- 4.04-4.17 (m, 2H), 4.59-4.64 (m, yl)piperidine-1-1H), 6.81-6.83 (m, 2H), 7.14 (s, carboxamide 1H), 7.22-7.26 (m, 1H),7.30-7.32 (m, 1H), 7.47-7.51 (m, 2H), 7.57 (s, 1H), 7.63-7.65 (m, 1H),7.98 (s, 1H), 8.09-8.11 (m, 2H) (4 exchangeable protons not observed) 83,5-dibromo-Na-{[4-(2- 11, 17  (400 MHz, MeOD) δ: ppm 1.26- m/z 882.5,oxo-2,3-dihydro-1H- Procedure 1 1.51 (m, 5H), 1.51-1.72 (m, 2H), 884.2(ES⁺), at imidazo[4,5-b]pyridin-1- 1.72-1.92 (m, 3H), 1.96-2.08 (m, 2.47min, 100% yl)piperidin-1- 1H), 2.08-2.38 (m, 2H), 2.79-3.05yl]carbonyl}-N-{(2S)-1- (m, 5H), 3.33-3.42 (m, 3H), 3.54-oxo-3-(piperidin-4-yl)-1- 3.64 (m, 1H), 3.64-3.79 (m, 3H),[4-(pyridin-4-yl)piperazin- 3.81-4.05 (m, 4H), 4.17 (d, J = 12.9,1-yl]propan-2-yl}-D- 2H), 4.35-4.45 (m, 1H), 7.07 (dd, tyrosinamide J =7.8, 5.5, 1H), 7.14 (d, J = 7.4, 2H), 7.42-7.52 (m, 3H), 7.95 (d, J =4.3, 1H), 8.14 (d, J = 7.4, 2H) 9 3,5-dibromo-Nα-{[4-(2- 12, 17  (400MHz, DMSO-d₆) δ: ppm 0.68- m/z 894.4, oxo-1,4- Procedure 1 0.78 (m, 1H),0.85-1.04 (m, 1H), 896.5, 898.4 dihydroquinazolin-3(2H)- 1.07-1.18 (m,1H), 1.21-1.39 (m, (ES⁺), at 2.26 yl)piperidin-1- 2H), 1.39-1.60 (m,5H), 1.60-1.79 and 2.47 min, yl]carbonyl}-N-{(2S)-1- (m, 2H), 1.86-2.14(m, 2H), 2.53- 100% oxo-3-(piperidin-4-yl)-1- 2.86 (m, 6H), 2.87-3.12(m, 4H), [4-(pyridin-4-yl)piperazin- 3.55-3.75 (m, 3H), 3.75-3.96 (m,1-yl]propan-2-yl}-D- 1H), 3.97-4.10 (m, 2H), 4.11-4.33 tyrosinamide (m,3H), 4.72-4.82 (m, 1H), 6.52 (t, J = 8.6, 1H), 6.78 (td, J = 7.4, 16.8,4H), 7.00-7.18 (m, 2H), 7.22-7.29 (m, 2H), 7.58-7.65 (m, 1H), 7.88 (d, J= 7.8, 1H), 7.99-8.06 (m, 1H), 8.07-8.25 (m, 2H), 9.16 (s, 1H) 10N-[(2R)-3-(7-methyl-1H- 13, 17  (400 MHz, DMSO-d₆) δ: ppm 1.12- m/z774.7 (ES⁻), indazol-5-yl)-1-oxo-1- Procedure 1 1.57 (m, 8H), 1.66 (d, J= 12.5, 1H), 776.6 (ES⁺), at ({(2S)-1-oxo-3-(piperidin- 1.85 (d, J =12.5, 1H), 2.52-2.78 (m, 3.62 min, 100% 4-yl)-1-[4-(pyridin-4- 4H), 2.89(t, J = 10.9, 1H), 2.95-3.06 yl)piperazin-1-yl]propan- (m, 1H),3.10-3.30 (m, 2H), 3.42- 2-yl}amino)propan-2-yl]- 3.54 (m, 1H), 3.61 (d,J = 5.1, 4H), 4-(2-oxo-1,4- 3.72-3.90 (m, 4H), 3.91-4.13 (m,dihydroquinazolin-3(2H)- 4H), 4.14-4.37 (m, 3H), 4.78-4.92yl)piperidine-1- (m, 2H), 6.65 (d, J = 7.8, 1H), 6.74 carboxamide (d, J= 7.8, 1H), 6.85 (t, J = 7.4, 1H), 7.03 (d, J = 7.0, 1H), 7.08-7.20 (m,4H), 7.45 (s, 1H), 7.98 (s, 1H), 8.27 (d, J = 5.9, 3H), 8.43 (d, J =8.6, 1H), 8.55 (d, J = 9.0, 1H), 9.21 (s, 1H), 13.53 (br s, 1H) 11N-[(2R)-3-(7-methyl-1H- 7, 17 (400 MHz, CD₃OD) δ: ppm 1.06- m/z 762.8(ES⁻), indazol-5-yl)-1-oxo-1- Procedure 1 1.20 (m, 4H), 1.22-1.32 (m,1H), 764.7 (ES⁺), at ({(2S)-1-oxo-3-(piperidin- 1.39-1.58 (m, 5H),1.76-1.83 (m, 3.41 min, 100% 4-yl)-1-[4-(pyridin-4- 2H), 1.84-1.95 (m,2H), 2.45-2.56 yl)piperazin-1-yl]propan- (m, 2H), 2.53 (s, 3H), 3.02(dd, 2-yl}amino)propan-2-yl]- J = 13.8, 9.2, 2H), 3.15-3.27 (m, 2H),2′-oxo-1′,2′-dihydro-1H- 3.38-3.42 (m, 1H), 3.48-3.59 (m,spiro[piperidine-4,4′- 3H), 3.59-3.74 (m, 2H), 3.83-3.99 pyrido[2,3- (m,4H), 4.63 (dd, J = 9.0, 6.8, 1H), d][1,3]oxazine]-1- 6.80-6.86 (m, 2H),7.06 (dd, J = 7.7, carboxamide 5.0, 1H), 7.12-7.21 (m, 2H), 7.48 (s,1H), 7.97 (s, 1H), 8.11-8.15 (m, 2H), 8.17-8.20 (m, 1H) (5 exchangeableprotons not observed) 12 N-[(2R)-1-({(2S)-3-{1-[2- 8, 22 (400 MHz,CD₃OD) δ: ppm 0.98- m/z 835.7 (dimethylamino)ethyl]pipe Procedure 1,1.22 (m, 3H), 1.37-1.51 (m, 3H), (ES+), at 3.19, ridin-4-yl}-1-oxo-1-[4-Step 1 1.60-1.77 (m, 6H), 2.00-2.09 (m, 95% (pyridin-4-yl)piperazin-1-1H), 2.17-2.26 (m, 2H), 2.30 (s, yl]propan-2-yl}amino)-3- 6H), 2.42-2.54(m, 7H), 2.78-2.93 (7-methyl-1H-indazol-5- (m, 4H), 3.02-3.08 (m, 1H),3.15- yl)-1-oxopropan-2-yl]-4- 3.20 (m, 1H), 3.31-3.42 (m, 1H),(2-oxo-2,3-dihydro-1H- 3.48-3.56 (m, 3H), 3.61-3.65 (m,imidazo[4,5-b]pyridin-1- 1H), 3.86-3.92 (m, 2H), 4.12-4.16yl)piperidine-1- (m, 2H), 4.37-4.44 (m, 1H), 4.60 (t, carboxamide J =7.8, 1H), 6.80-6.85 (m, 2H), 6.97 (dd, J = 7.8, 5.1, 1H), 7.16 (s, 1H),7.34 (dd, J = 7.8, 1.2, 1H), 7.49 (s, 1H), 7.92 (dd, J = 5.1, 1.2, 1H),7.97 (s, 1H), 8.08-8.19 (m, 2H) (4 exchangeable protons not observed) 133-(4-{(2S)-2-{[(2R)-3-(7- 8, 23 (400 MHz, CD₃OD) δ: ppm 0.57- m/z 849.8methyl-1H-indazol-5-yl)- Procedure 3 0.73 (m, 2H), 0.86-1.10 (m, 4H),(ES+), at 1.82, 2-({[4-(2-oxo-2,3-dihydro- 1.21-1.40 (m, 2H), 2.16 (s,3H), 95% 1H-imidazo[4,5-b]pyridin- 2.26-2.44 (m, 4H), 2.52-2.67 (m,1-yl)piperidin-1- 2H), 2.74-2.95 (m, 3H), 3.03-3.41yl]carbonyl}amino)propanoyl]amino}- (m, 7H), 3.46-3.62 (m, 6H), 3.79-3-oxo-3-[4- 4.06 (m, 4H), 6.32-6.36 (m, 2H), (pyridin-4-yl)piperazin-1-6.56-6.59 (m, 1H), 6.68-6.83 (m, yl]propyl}piperidin-1-yl)- 2H), 7.15(br s, 1H), 7.68 (br s, 3-oxopropanoic acid, 1H), 7.69-7.74 (m, 2H),7.90-7.93 ammonium salt (m, 1H) (8 exchangeable protons not observed) 143,5-dibromo-Na-[(2′-oxo- 14, 17  (400 MHz, CD₃OD) δ: ppm 0.96- m/z 884.5(ES⁺), 1′,2′-dihydro-1H- Procedure 1 1.20 (m, 5H), 1.23-1.48 (m, 3H), at2.27 min, spiro[piperidine-4,4′- 1.58 (ddd, J = 14.3, 9.6, 4.3, 1H),100% pyrido[2,3-d][1,3]oxazin]- 1.65-1.74 (m, 1H), 1.80-2.06 (m,1-yl)carbonyl]-N-{(2S)-1- 5H), 2.62 (td, J = 12.7, 3.1, 1H),oxo-3-(piperidin-4-yl)-1- 2.77-2.99 (m, 3H), 3.45-3.64 (m,[4-(pyridin-4-yl)piperazin- 4H), 3.81-4.04 (m, 4H), 4.48 (dd,1-yl]propan-2-yl}-D- J = 9.4, 7.0, 1H), 6.82-6.85 (m, 2H), tyrosinamide7.08 (dd, J = 7.6, 4.9, 1H), 7.28 (s, 2H), 7.34 (s, 1H), 7.56 (dd, J =7.6, 1.8, 1H), 8.11-8.20 (m, 3H) (2 protons obscured by solvent peaks, 5exchangeable protons not observed)Biological and Biophysical Methods

Cloning, Baculovirus generation, large-scale infection of Sf21 cells andmembrane preparation. Human Calcitonin Receptor Like Receptor (CRLR) andhuman RAMP1 were cloned into Invitrogen's (ThermoFisher Scientific, UK)pFastBac dual expression vector. Transposition of CRLR/RAMP1 DNA wasperformed using Invitrogen's Bac-to-Bac Baculovirus Expression Systems.P0 baculovirus was generated by transfecting SF9 cells with bacmid DNAusing Cellfectin® II transfection reagent (ThermoFisher Scientific, UK,catalog number 10362-100). Following P0 generation P1 virus was thengenerated ready for large scale infection and membrane preparation. Sf21cells were grown in expression medium ESF921 (Expression Systems, USA,catalog number 96-001-01) supplemented with 10% heat-inactivated FBS and1% Pen/Strep and were infected at a cell density of 2.5×10⁶ cells/mL andan MOI of 2. Expression was carried out over 48 h in a shaking incubatorset at 27° C. The cell culture was centrifuged at 2,500 rcf for 10 minat 4° C. The pellets were resuspended in cold PBS supplemented withRoche's Complete EDTA-free protease inhibitor cocktail tablets (RocheApplied Sciences, catalog number 05056489001), 1 mM PMSF and 1 mM EDTA.The resuspended cell paste was then centrifuged at 3,273 rcf for 12 minat 4° C. The supernatant was discarded and the pellet frozen at −80° C.The cell pellet from a 4 L culture was resuspended in buffer containing50 mM Hepes pH 7.5, 150 mM NaCl, 8 Roche EDTA-free protease inhibitorcocktail tablets and 1 mM PMSF. The suspension was left stirring at rtfor 1 h and then homogenised for 90 s at 9,500 rpm using a VDI 25 (VWR,USA) homogeniser. The cells were then lysed using a Microfluidizerprocessor M-110L Pneumatic (Microfluidics, USA). After lysis, themixture was homogenised for 90 s at 9,500 rpm and then centrifuged at335 rcf for 10 min. The supernatant was then further ultra-centrifugedat 42,000 rpm for 90 min. After ultra-centrifugation, the supernatantwas discarded and the pellet was resuspended in 50 mL (25 mL for each 2L culture) of buffer containing 50 mM Hepes pH 7.5, 150 mM NaCl, 3 RocheEDTA-free protease inhibitor cocktail tablets and 1 mM PMSF. Thesuspension was then homogenised for 90 s at 9,500 rpm. The resultingmembranes were then stored at −80° C.

Radioligand binding assay. Human CGRP receptors expressed (consisting ofCRLR and RAMP1) in insect Sf21 cell membrane homogenates werere-suspended in the binding buffer (10 mM HEPES, pH 7.4, 5 mM MgCl₂,0.2% BSA) to a final assay concentration of 0.6 μg protein per well.Saturation isotherms were determined by the addition of variousconcentrations of ³H-telcagepant (Ho et al, The Lancet, 2008, 372, 2115)(in a total reaction volume of 250 μL) for 60 min at rt. At the end ofthe incubation, membranes were filtered onto a unifilter, a 96-wellwhite microplate with bonded GF/B filter pre-incubated with 0.5% PEI,with a Tomtec cell harvester and washed 5 times with distilled water.Non-specific binding (NSB) was measured in the presence of 10 nM MK-3207hydrochloride (CAS No. 957116-20-0). Radioactivity on the filter wascounted (1 min) on a microbeta counter after addition of 50 μL ofscintillation fluid. For inhibition experiments, membranes wereincubated with 0.5 nM ³H-telcagepant and 10 concentrations of theinhibitory compound (0.001-10 μM). IC₅₀ values were derived from theinhibition curve and the affinity constant (K_(i)) values werecalculated using the Cheng-Prussoff equation (Cheng et al, Biochem.Pharmacol. 1973, 22, 3099-3108). The pK_(i) values (where pK_(i)=−log₁₀K_(i)) of certain compounds of the invention are tabulated below.

cAMP functional assay. cAMP production following receptor activation wasdetermined using the Homogeneous Time-Resolved Fluorescence (HTRF) cAMPdynamic-2 assay (Cisbio, France). The human neuroblastoma cell lineSK-N-MC endogenously expressing the human CGRP receptor was seeded at adensity of 12,500 cells/well in solid walled 96 well half area plates(Costar, Catalog Number 3688, Corning Life Sciences, Germany). After 16h incubation at 37° C. media was removed and cells were incubated at 37°C. for 30 min in serum free media containing 500 μM IBMX (Tocris,Abingdon, UK, Catalog Number 2845) and increasing concentrations of testantagonist. Following this cells were challenged with an EC₈₀concentration of human CGRP (0.3 nM) for a further 30 min at 37° C. andthen cAMP production was determined as manufacturer's instructionsbefore plates were read on a PheraStar fluorescence plate reader (BMGLabTech, Germany). IC₅₀ values were derived from the inhibition curve.The pIC₅₀ values (where pIC₅₀=−log₁₀IC₅₀) were converted to a functionalpK_(b) value using a modified Cheng-Prussoff equation whereK_(d)=agonist EC₅₀ and L hot=agonist challenge concentration. The pK_(b)values of certain compounds of the invention are detailed in Table 3.

TABLE 3 Ex pK_(i) pK_(b) No. Name Structure average average  1N-[(2R)-3-(7-methyl-1H-indazol- 5-yl)-1-oxo-1-({(2S)-1-oxo-3-(piperidin-4-yl)-1-[4-(pyridin-4- yl)piperazin-1-yl]propan-2-yl}amino)propan-2-yl]-4-(2-oxo- 2,3-dihydro-1H-imidazo[4,5-b]pyridin-1-yl)piperidine-1- carboxamide

10.7 11.2  2 tert-butyl 4-{(2S)-2-{[(2R)-3-(7-methyl-1H-indazol-5-yl)-2-({[4- (2-oxo-1,2-dihydroquinolin-3-yl)piperidin-1-yl]carbonyl}amino) propanoyl]amino}-3-oxo-3-[4-(pyridin-4-yl)piperazin-1-yl] propyl}piperidine-1- carboxylate

10.0 9.7  3 N-[(2R)-3-(7-methyl-1H-indazol-5-yl)-1-oxo-1-({(2S)-1-oxo-3- (piperidin-4-yl)-1-[4-(pyridin-4-yl)piperazin-1-yl]propan-2-yl} amino)propan-2-yl]-4-(2-oxo-1,2-dihydroquinolin-3-yl) piperidine-1-carboxamide

10.1 9.8  4 N-[(2R)-3-(7-methyl-1H-indazol-5-yl)-1-oxo-1-({(2S)-1-oxo-3-(1- propylpiperidin-4-yl)-1-[4-(pyridin-4-yl)piperazin-1- yl]propan-2-yl}amino)propan-2-yl]-4-(2-oxo-1,2-dihydroquinolin- 3-yl)piperidine-1-carboxamide

10.4 10.7  5 3,5-dibromo-Nα-{[4-(2-oxo-1,2-dihydroquinolin-3-yl)piperidin-1- yl]carbonyl}-N-{(2S)-1-oxo-3-(piperidin-4-yl)-1-[4-(pyridin-4- yl)piperazin-1-yl]propan-2-yl]-D-tyrosinamide

10.2 9.6  6 N-[(2R)-3-(7-methyl-1H-indazol-5-yl)-1-oxo-1-({(2S)-1-oxo-3-(1- pentanoylpiperidin-4-yl)-1-[4-(pyridin-4-yl)piperazin-1-yl] propan-2-yl}amino)propan-2-yl]-4-(2-oxo-1,2-dihydroquinolin- 3-yl)piperidine-1-carboxamide

10.0 10.0  7 N-[(2R)-1-({(2S)-3-(1- ethylpiperidin-4-yl)-1-oxo-1-[4-(pyridin-4-yl)piperazin-1-yl] propan-2-yl}amino)-3-(7-methyl-1H-indazol-5-yl)-1-oxopropan-2- yl]-4-(2-oxo-1,2-dihydroquinolin-3-yl)piperidine-1-carboxamide

10.4 10.6  8 3,5-dibromo-Nα-{[4-(2-oxo-2,3- dihydro-1H-imidazo[4,5-b]pyridin-1-yl)piperidin-1-yl] carbonyl}-N-{(2S)-1-oxo-3-(piperidin-4-yl)-1-[4-(pyridin-4- yl)piperazin-1-yl]propan-2-yl}-D-tyrosinamide

10.4 10.8  9 3,5-dibromo-Nα-{[4-(2-oxo-1,4- dihydroquinazolin-3(2H)-yl)piperidin-1-yl]carbonyl}-N- {(2S)-1-oxo-3-(piperidin-4-yl)-1-[4-(pyridin-4-yl)piperazin-1-yl] propan-2-yl}-D-tyrosinamide

10.1 11.2 10 N-[(2R)-3-(7-methyl-1H-indazol- 5-yl)-1-oxo-1-({(2S)(2S)-1-oxo-3- (piperidin-4-yl)-1-[4-(pyridin-4-yl)piperazin-1-yl]propan-2- yl}amino)propan-2-yl]-4-(2-oxo-1,4-dihydroquinazolin-3(2H)- yl)piperidine-1-carboxamide

10.2 10.1 11 N-[(2R)-3-(7-methyl-1H-indazol-5-yl)-1-oxo-1-({(2S)-1-oxo-3- (piperidin-4-yl)-1-[4-(pyridin-4-yl)piperazin-1-yl]propan-2-yl} amino)propan-2-yl]-2′-oxo-1′,2′-dihydro-1H-spiro[piperidine- 4,4′-pyrido[2,3-d][1,3]oxazine]-1-carboxamide

10.5 10.3 12 N-[(2R)-1-({(2S)-3-{l-[2- (dimethylamino)ethyl]piperidin-4-yl}-1-oxo-1-[4-(pyridin-4- yl)piperazin-1-yl]propan-2-yl}amino)-3-(7-methyl-1H- indazol-5-yl)-1-oxopropan-2-yl]-4-(2-oxo-2,3-dihydro-1H-imidazo [4,5-b]pyridin-1-yl)piperidine-l-carboxamide

10.6 10.6 13 3-(4-{(2S)-2-{[(2R)-3-(7-methyl-1H-indazol-5-yl)-2-({[4-(2-oxo- 2,3-dihydro-1H-imidazo[4,5-b]pyridin-1-yl)piperidin-1-yl] carbonyl}amino)propanoyl]amino}-3-oxo-3-[4-(pyridin- 4-yl)piperazin-1-yl]propyl}piperidin-1-yl)-3-oxo- propanoic acid, ammonium salt

10.0 9.9 14 3,5-dibromo-Nα-[(2′-oxo-1′,2′-dihydro-1H-spiro[piperidine-4,4′- pyrido[2,3-d][1,3]oxazin]-1-yl)carbonyl]-N-{(2S)-1-oxo-3- (piperidin-4-yl)-1-[4-(pyridin-4-yl)piperazin-1-yl]propan-2-yl}- D-tyrosinamide

10.1 10.8

Receptor Kinetic Profiling. It is appreciated that the kinetic profileof a small molecule at the relevant biological target can have an impactupon the pharmacodynamic effect of the molecule in vivo (Copeland,Expert Opin. Drug Discov., 2010, 5, 305). For example, olcegepant hasslow kinetics at the CGRP receptor (Schindler, Doods, Eur. J.Pharmacol., 2002, 442, 187), a factor which may contribute to itsprolonged efficacy in migraine treatment in humans (47% headache-freerate at 24 h after intravenous infusion of a 2.5 mg dose; Olesen et al,N. Eng. J. Med., 2004, 350, 1104). In a similar way, MK-3207 has alsobeen shown to demonstrate relatively slow dissociation for the CGRPreceptor (Salvatore et al, J. Pharmacol. Exp. Ther., 2010, 333, 152).The CGRP receptor kinetics of compounds of the invention and referenceCGRP receptor antagonists have been profiled using the surface plasmonresonance technique below, and are detailed in Table 4.

Kinetic analyses were run on a Biacore T200 instrument (GE HealthcareBio-Sciences AB, Uppsala, Sweden) at 25° C. using 0.05 mM EDTA, PBS (10mM phosphate buffer, 2.7 mM KCl, 137 mM NaCl) pH 7.4, 0.005% v/vSurfactant P20, 5% DMSO as the running buffer. The purified CGRPreceptor ectodomain complex containing a hexa-His tag (Moore et al,Structure, 2010, 18, 1083-1093) was immobilised on a sensor chip NTA (GEHealthcare Bio-Sciences AB) by the capture-couple technique (Rich et al,Anal. Biochem., 2011, 409, 267-272). The chip was loaded with Ni²⁺ andcarboxyl groups of the dextran matrix were activated by EDC/NHS. Thereceptor ectodomain complex (100 nM in running buffer) was then injectedand immobilised via the His-tag and amino groups. Two fold dilutionseries of each compound (five concentrations, in the range 25-40 nM)were injected. Blank-subtracted data were fitted to a 1:1 interactionmodel to obtain kinetic parameters which are expressed in Table 4 asdissociation half-life (t_(1/2)=(In 2/off-rate (k_(d)))/60).

The data presented indicates that each of the examples 2, 4, 6, 7, 8, 9,11, 12 and 14 have the property of slow receptor dissociation that iscomparable or slower in off-rate than olcegepant or MK-3207.

TABLE 4 Reference Compound/ Example CGRP t_(1/2) (min) olcegepant 30MK-3207 27 telcagepant 2 BMS-927711 2 Example 1 5 Example 2 78 Example 34 Example 4 39 Example 5 12 Example 6 20 Example 7 36 Example 8 95Example 9 32 Example 10 3 Example 11 65 Example 12 101 Example 13 Notdetermined Example 14 38

Pharmacokinetic profiling. The pharmacokinetic profiles of Examples andreference compounds have been assessed in male Sprague Dawley® rats viaintravenous (iv), sub-cutaneous (sc) and intranasal (IN) routes ofdelivery, and in male Cynomolgus Monkeys via iv and sc routes ofdelivery. Pharmacokinetic data for Examples of the invention and areference compound, olcegepant, are detailed in Tables 5 and 6.

Methods: For rat studies, groups of three male Sprague Dawley® rats,typically ranging in weight between 180 and 300 g, were given a singledose of Example or reference compound via one of the following routes:iv, sc or IN, using doses, dose volumes and vehicles specified in Table5. Prior to IN dosing rats were anaesthetised with an intramuscular doseof 25-30 mg/kg ketamine cocktail (ketamine, xylazine hydrochloride andacepromazine maleate in saline) and the dose is introduced over 20-30 svia a polyethylene PE-10 tube inserted approximately 5 mm into the nasalcavity of the rat.

For cynomolgus monkey studies, groups of three male monkeys, typicallyranging in weight between 3.0 and 4.5 kg, were given a single dose ofExample or reference compound via one of the following routes: iv or sc,using doses, dose volumes and vehicles specified in Table 5. Followingdosing by the routes above blood samples were taken at several timepoints (typically pre-dose, 0.083, 0.25, 0.5 1, 2, 4, 8 and 24 h) viaserial tail vein bleeds (rat) or cephalic or saphenous vein (monkey)from the animal and centrifuged to separate plasma for analysis byLC/MS/MS assay. WinNonlin v6.2 statistics software (PharsightCorporation, California, USA) was used to generate pharmacokineticparameters using the non-compartmental model.

TABLE 5 Rat iv pharmacokinetics Dose Dose volume Clearance (mg/kg)(mL/kg) Vehicle (mL/min/kg) olcegepant 5 1 10% DMAC + 10% 18SolutolHS15 + 80% Saline Example 11 2 1 10% DMAC + 10% 17 SolutolHS15 +80% Saline Example 14 2 1 10% DMAC + 10% 22 SolutolHS15 + 80% Saline Ratsc pharmacokinetics Bioavail- Dose Dose volume ability (mg/kg) (mL/kg)Vehicle (%) olcegepant 1 5 10% DMAC + 10% 48% SolutolHS15 + 80% SalineExample 11 1 2 Acidified saline 100%  Example 14 1 2 Acidified saline96% Rat IN pharmacokinetics Dose Bioavail- Dose concentration, ability(mg/kg) Dose volume Vehicle (%) olcegepant 1.3 6 mg/mL, 50 μL Acidifiedsaline 8 Example 11 1 12 mg/mL, 25 μL Acidified saline 40 Example 14 112 mg/mL, 25 μL Acidified saline 19

TABLE 6 Cynomolgus monkey iv pharmacokinetics Dose Dose volume Clearance(mg/kg) (mL/kg) Vehicle (mL/min/kg) Example 11 0.5 1 Acidified saline 2Example 14 0.4 0.5 Acidified saline 3 Cynomolgus monkey scpharmacokinetics Dose Dose volume Bioavailability (mg/kg) (mL/kg)Vehicle (%) Example 11 0.5 1 Acidified saline 100 Example 14 Not tested

Thermodynamic solubility profiling. A 50 mM DMSO stock solution of testcompound was prepared, and from this, a working solution of 1 mM wasprepared by dilution with DMSO. The UV absorbance of working solutionwas scanned from 220 nm to 1000 nm to identify the wavelength maxima oftest compound. The 1 mM working solution was then serially diluted inDMSO to different concentrations to determine linearity/calibrationcurve. To ascertain the aqueous thermodynamic solubility of testcompound, samples were added to a volume of PBS buffer (pH 7.4) orSodium Phosphate Buffer (pH 6.0) which was appropriate to generate afinal concentration of 1 mg/mL if all test compound dissolved. Theresulting solution was then kept on a RotoSpin shaker at 50 rpm for 24 hat rt before the solution was filtered using 0.45 micron PVDF injectorfilters in order to remove the insoluble fraction of the compound.Subsequently, 150 uL of the filtrate is taken for quantification using aUV spectrophotometer, acquiring the optical density of standardsolutions and test compound at the same wavelength maxima. From theoptical density of test compound the thermodynamic solubility iscalculated using the linearity/calibration curve and expressed asmicromolar (μM). Solubility profiles of certain compounds of theinvention are detailed in Table 7.

TABLE 7 Reference Cpd/ Thermodynamic solubility (μM) Example pH 6 pH 7.4olcegepant  150 431 Example 1 3387 3222  Example 2 Not tested Not testedExample 3  10 509 Example 4 Not tested Not tested Example 5   9 113Example 6 Not tested Not tested Example 7 Not tested Not tested Example8 1029 890 Example 9 Not tested Not tested Example 10 1171 1205  Example11 1263 1426  Example 12 1648 1955  Example 13 Not tested Not testedExample 14 1111 862

The invention claimed is:
 1. A compound of formula (I)

or a salt thereof, wherein R¹ is H or Q-(C₁-C₆)alkyl; where Q is a bond,C(O) or C(O)O and where the (C₁-C₆)alkyl can be optionally substitutedby N(C₁-C₃alkyl)₂ or CO₂H; R² is H or forms a spirocyclic heterocyclicring with R³; R³ forms a spirocyclic heterocyclic ring with R² or is aheterocyclic ring if R² is H; and R⁴ is an optionally substituted arylgroup which is fused to a further ring.
 2. The compound according toclaim 1, or a salt thereof, wherein R⁴ is


3. The compound according to claim 1, or a salt thereof, wherein R² is Hand R³ is:


4. The compound according to claim 1, or a salt thereof, wherein R²forms a spirocyclic heterocyclic ring with R³ to form:


5. The compound according to claim 1, or a salt thereof, wherein R² is Hor forms a spirocyclic heterocyclic ring with R³ to form:

and wherein when R² is H, R³ is:

and R⁴ is:


6. The compound according to claim 1, or a salt thereof, wherein R¹ isH, CO₂ ^(t)Bu, CH₂CH₃, CH₂CH₂CH₃, COCH₂CH₂CH₂CH₃, CH₂CH₂N(CH₃)₂,COCH₂CO₂H.
 7. The compound according to claim 1, or a salt thereof,wherein R¹ is H.
 8. The compound according to claim 1, wherein thecompound is selected from the group consisting of:N-[(2R)-3-(7-methyl-1H-indazol-5-yl)-1-oxo-1-({(2S)-1-oxo-3-(piperidin-4-yl)-1-[4-(pyridin-4-yl)piperazin-1-yl]propan-2-yl}amino)propan-2-yl]-4-(2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-1-yl)piperidine-1-carboxamide;tert-butyl4-{(2S)-2-{[(2R)-3-(7-methyl-1H-indazol-5-yl)-2-({[4-(2-oxo-1,2-dihydroquinolin-3-yl)piperidin-1-yl]carbonyl}amino)propanoyl]amino}-3-oxo-3-[4-(pyridin-4-yl)piperazin-1-yl]propyl}piperidine-1-carboxylate;N-[(2R)-3-(7-methyl-1H-indazol-5-yl)-1-oxo-1-({(2S)-1-oxo-3-(piperidin-4-yl)-1-[4-(pyridin-4-yl)piperazin-1-yl]propan-2-yl}amino)propan-2-yl]-4-(2-oxo-1,2-dihydroquinolin-3-yl)piperidine-1-carboxamide;N-[(2R)-3-(7-methyl-1H-indazol-5-yl)-1-oxo-1-({(2S)-1-oxo-3-(1-propylpiperidin-4-yl)-1-[4-(pyridin-4-yl)piperazin-1-yl]propan-2-yl}amino)propan-2-yl]-4-(2-oxo-1,2-dihydroquinolin-3-yl)piperidine-1-carboxamide;N-[(2R)-3-(7-methyl-1H-indazol-5-yl)-1-oxo-1-({(2S)-1-oxo-3-(1-pentanoylpiperidin-4-yl)-1-[4-(pyridin-4-yl)piperazin-1-yl]propan-2-yl}amino)propan-2-yl]-4-(2-oxo-1,2-dihydroquinolin-3-yl)piperidine-1-carboxamide;N-[(2R)-1-({(2S)-3-(1-ethylpiperidin-4-yl)-1-oxo-1-[4-(pyridin-4-yl)piperazin-1-yl]propan-2-yl}amino)-3-(7-methyl-1H-indazol-5-yl)-1-oxopropan-2-yl]-4-(2-oxo-1,2-dihydroquinolin-3-yl)piperidine-1-carboxamide;N-[(2R)-3-(7-methyl-1H-indazol-5-yl)-1-oxo-1-({(2S)-1-oxo-3-(piperidin-4-yl)-1-[4-(pyridin-4-yl)piperazin-1-yl]propan-2-yl}amino)propan-2-yl]-4-(2-oxo-1,4-dihydroquinazolin-3(2H)-yl)piperidine-1-carboxamide;N-[(2R)-3-(7-methyl-1H-indazol-5-yl)-1-oxo-1-({(2S)-1-oxo-3-(piperidin-4-yl)-1-[4-(pyridin-4-yl)piperazin-1-yl]propan-2-yl}amino)propan-2-yl]-2′-oxo-1′,2′-dihydro-1H-spiro[piperidine-4,4′-pyrido[2,3-d][1,3]oxazine]-1-carboxamide;N-[(2R)-1-({(2S)-3-{1-[2-(dimethylamino)ethyl]piperidin-4-yl}-1-oxo-1-[4-(pyridin-4-yl)piperazin-1-yl]propan-2-yl}amino)-3-(7-methyl-1H-indazol-5-yl)-1-oxopropan-2-yl]-4-(2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-1-yl)piperidine-1-carboxamide;and3-(4-{(2S)-2-{[(2R)-3-(7-methyl-1H-indazol-5-yl)-2-({[4-(2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-1-yl)piperidin-1-yl]carbonyl}amino)propanoyl]amino}-3-oxo-3-[4-(pyridin-4-yl)piperazin-1-yl]propyl}piperidin-1-yl)-3-oxopropanoicacid, ammonium salt; or a salt thereof.
 9. The compound according toclaim 8, wherein the compound is:

or a salt thereof.
 10. A method of synthesis of the compound accordingto claim 1 comprising any one of the following procedures: